Primary gastric Merkel cell carcinoma harboring DNA polyomavirus

Human Pathology (2014) xx, xxx–xxx

www.elsevier.com/locate/humpath

Case study

Primary gastric Merkel cell carcinoma harboring DNA polyomavirus. First description of an unusual high-grade neuroendocrine carcinoma Carlo Capella MD a , Alessandro Marando MD a , Erika Longhi PhD b , Barbara Bernasconi BD a , Giovanna Finzi PhD c , Carlo Parravicini MD d , Fausto Sessa MD a , Stefano La Rosa MD c,⁎ a

Department of Surgical and Morphological Sciences, University of Insubria, 21100 Varese, Italy Department of Clinical Sciences, University of Milan, 20157 Milan, Italy c Department of Pathology, Ospedale di Circolo, 21100 Varese, Italy d Unit of Pathology, “L. Sacco” University Hospital, 20157 Milan, Italy b

Received 20 December 2013; accepted 16 January 2014

Keywords: Merkel cell carcinoma; Stomach; Neuroendocrine carcinoma; Merkel cell polyomavirus

Summary Merkel cell carcinoma (MCC) is a skin cancer that can also rarely arise in extracutaneous sites including mucosal surfaces. About 80% of MCCs harbor the Merkel cell polyomavirus (MCPyV). All cases of gastric MCCs so far reported were metastases from cutaneous sources. In the present article, we describe for the first time a primary gastric MCC harboring MCPyV. A 72-yearold man presented to clinical observation due to epigastric pain. Upper endoscopy revealed an ulcerated gastric tumor. The patient underwent total gastrectomy. The tumor was composed of mitotically active monomorphic small cells showing round nuclei with finely dispersed chromatin arranged in sheets and nests with large areas of necrosis. Tumor cells were positive for neuroendocrine markers and showed paranuclear dot immunoreactivity for cytokeratin 20. MCPyV was demonstrated with immunohistochemistry and electron microscopy, which showed intranuclear and intracytoplasmic viral particles. The MCPyV DNA in tumor cells was demonstrated with polymerase chain reaction analysis. © 2014 Published by Elsevier Inc.

1. Introduction

☆ Competing interests: None. ⁎ Corresponding author. Department of Pathology, Ospedale di Circolo, viale Borri 57, 21100 Varese Italy. E-mail address: [email protected] (S. La Rosa).

0046-8177/$ – see front matter © 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.humpath.2014.01.019

Merkel cell carcinoma (MCC) is a relatively rare and aggressive skin cancer that can also arise in extracutaneous sites and can potentially metastasize to all organs of the human body [1]. Exceptional primary MCCs arising in mucosal surfaces have been described, but cases of gastric MCC so far reported in the literature were metastases from cutaneous sources. In the present article, we describe a

2 primary MCC of the stomach harboring Merkel cell polyomavirus (MCPyV), which has been recently detected also in other extracutaneous MCCs [7].

2. Materials and methods 2.1. Case history A 72-year-old man with a long history of chronic obstructive pulmonary disease and hypertension presented to clinical observation due to recent onset of increasing epigastric pain and 20-lb weight loss over the previous month. On examination, the patient was normotensive, and pallor of the conjunctiva and mild epigastric tenderness were seen. Laboratory results were significant for hemoglobin level of 8.5 g/dL and hematocrit of 28.1. Upper endoscopy revealed a large ulcerated mass extending from the cardias to the body of the stomach. A biopsy was performed, and histologic examination revealed sheets and groups of small lymphocyte-like neoplastic cells immunoreactive for pancytokeratin and chromogranin A consistent with a poorly differentiated neuroendocrine carcinoma. The patient underwent total gastrectomy and splenectomy. After the postoperative pathological diagnosis, the patient, questioned about history of cutaneous lesions, denied the presence of cancerous lesions of the skin. A complete and accurate examination of his skin showed no suspicious lesions. A postoperative F18 fluorodeoxyglucose positron emission tomography performed 2 months later showed accumulation of the tracer in the right external iliac fossa, near the urinary bladder, consistent with a bulky metastatic lymph node or with a peritoneal neoplastic localization. An abdominal computed tomographic scan confirmed the presence of the metastatic lymphoadenopathy in the right iliac fossa. Salvage chemotherapy based on etoposide and cisplatin was initiated, but the patient died 2 months later. Autopsy was not performed.

2.2. Histology, immunohistochemistry, and electron microscopy Routine hematoxylin and eosin–stained sections were prepared using a standard technique from formalin-fixed and paraffin-embedded (FFPE) blocks. Immunohistochemical analyses, including a panel of monoclonal and polyclonal antibodies (Table), was performed on 3-μm-thick sections cut from selected paraffin blocks using the Ventana Medical System Benchmark XT Ultra (Ventana, Tucson, AZ). For electron microscopy, a piece of paraffin-embedded gastric MCC was used. The material was deparaffinized in chloroform, rehydrated, and washed in a 0.1 M sodium cacodylate buffer. Tissue fragments were postfixed in osmium and dehydrated and embedded in Epon-Araldite. Ultrathin sections contrasted with uranyl acetate and lead citrate were

C. Capella et al. Table

Antibodies and antisera used

Antibodies/ Antisera

P/M (Clone) Dilution Source

Synaptophysin M (snp88)

1:100

Chromogranin A Cytokeratin 20 Cytokeratin 7

1:1

BioGenex Laboratories, San Ramon, CA Ventana

1:100 1:200

Dako, Carpinteria, CA Dako

TdT PAX 5

M (LK2H10) M (K520.8) M (OV-TL 12/30) P P

1:1 1:2000

p63

M (4A4)

1:2

Claudin 18

1:300

PDX1

M (34H14L15) M (124) M (8G7G3/1) P

Ventana Santa Cruz Biotechnology, Santa Cruz, CA Cell Marque, Roklin, CA Invitrogen, Carlsbad, CA Dako Ventana

MCPyV

M (CM2B4) 1:100

p53 CD117 Ki-67

M (D07) P M (MIB1)

BCL2 TTF1

1:40 1:2 1:200

1:500 1:100 1:100

Santa Cruz Biotechnology Santa Cruz Biotechnology Dako Dako Dako

Abbreviation: P/M, polyclonal/monoclonal.

examined in a Morgagni electron microscope (Philips, Eindoven, the Netherlands).

2.3. Polyomavirus DNA detection Total DNA was extracted from FFPE tissue sections using the Qiagen Tissue Kit (Qiagen, Valencia, Spain) according to the manufacturer's instructions. DNA concentration and purity were determined by spectrometry. The maximum length of amplifiable DNA was assessed using multiplex polymerase chain reaction (PCR) on fragments of housekeeping genes of various sizes (100, 200, 300, 400, and 600 base pairs [bp]). The presence of MCPyV DNA was investigated using a PCR assay amplifying a 177-bp fragment targeted on the T-Ag gene [8]. Paraffin sections of nonneoplastic gastric tissue were used as a negative control, whereas DNA from a case of cutaneous MCC was used as a positive control.

2.4. Fluorescence in situ hybridization Fluorescence in situ hybridization (FISH) analysis was performed on FFPE sections following the method previously reported [9]. Split-signal probes for MYC, provided by

Primary gastric MCC harboring DNA polyomavirus Dakocytomation (Glostrup, Denmark) was used. The analysis was performed using direct viewing on a standard fluorescence microscope (Leica DMRA, Wetzlar, Germany) at ×100 magnification. FISH evaluation was performed according to a specific laboratory threshold value.

3. Results 3.1. Pathological findings The resected stomach contained a Borrmann type 3 carcinoma, measuring 12 cm in maximum diameter and involving the fundus and the cardias. Histologically, the tumor displayed a neuroendocrine architecture, with sheets and nests of monomorphic small cells showing round nuclei with finely dispersed chromatin encircled by a wellevident nuclear membrane (Fig. 1A and B). The tumor was mitotically highly active (mitotic count N10 per highpower field) and contained abundant areas of necrosis. Practically all tumor cells were positive for chromogranin A (Fig. 1C), synaptophysin, and BCL2. Moreover, about 90% of cells showed a paranuclear dot immunoreactive for cytokeratin 20 (Fig. 1D) and nuclear immunoreactivity for the anti-MCPyV antibody (CM2B4; Fig. 2A). Tumor cells were also positive for CD117 and p53. No immunoreac-

3 tivity for TTF1, cytokeratin 7, TdT, PAX5, p63, and claudin 18 was found. A nuclear positivity for PDX1 was detected in about 20% of neoplastic cells, and the Ki-67 labeling index was 60%. The neoplasm infiltrated the gastric wall including the subserosa, and metastases were found in 4 of 24 perigastric lymph nodes. The spleen did not show tumor infiltration. Electron microscopy revealed both intranuclear and rare intracytoplasmic viral particles measuring approximately 30 nm that were consistent with viral polyoma particles (Fig. 2B).

3.2. PCR analysis The multiplex PCR for housekeeping genes yielded the amplification of fragments of at least 300 bp on the DNA from the gastric MCC and from controls. The PCR specific for the MCPyV T-Ag gene was positive in the gastric MCC, with an amplified fragment of the same size of the amplicon generated on the positive control, whereas the DNA from the nonneoplastic gastric tissue was negative (Fig. 2C).

3.3. FISH analysis No MYC rearrangements were found with FISH using a split-signal probe for the MYC gene.

Fig. 1 The gastric MCC was characterized by sheets (A) of monomorphic small cells showing round nuclei with finely dispersed chromatin encircled by a well-evident nuclear membrane (B). A, Gastric mucosa is evident in the upper left corner, whereas carcinoma is well evident in the right part. Carcinomatous cells were strongly positive for chromogranin A (C) and showed a characteristic paranuclear dot immunoreactivity for cytokeratin 20 (D).

4

C. Capella et al.

Fig. 2 Tumor cells contained MCPyV, as demonstrated by immunohistochemistry using the anti-MCpyV antibody (A) and electron microscopy that showed intranuclear viral particles measuring approximately 30 nm (B). C, PCR analysis demonstrated the presence of MCPyV T-Ag gene. Lanes 1, 2, and 3 correspond to gastric MCC; lane 4, nonneoplastic tissue; and lane 5, cutaneous MCC used as a positive control.

4. Discussion The case herein reported suggests at least 3 interesting points of discussion: (1) the apparently primary origin of the MCC in the stomach, (2) the presence of MCPyV within neoplastic cells, and (3) the differential diagnosis between MCC and poorly differentiated neuroendocrine carcinomas (PD-NECs) from different anatomical sites. Although MCC most commonly develops in the skin, extracutaneous primary MCCs have been described [7]. However, MCCs of the gastrointestinal tract so far reported in the literature were metastases from cutaneous primary MCCs [2-6]. Although in the present case, the possibility of a metastasis from an occult cutaneous MCC undergoing spontaneous regression [10] cannot be excluded, several findings favor the primary origin in the stomach. The patient had no remarkable history of skin neoplasms and the result from the dermatologic examination was negative, as were results from the F18 fluorodeoxyglucose positron emission tomography and computed tomographic scan that showed accumulation of the tracer and tumor masses, respectively, only in the stomach and in abdominal lymph nodes.

Carcinomas identical to MCC of the skin, in terms of histology, immunohistochemistry, electron microscopy, and the presence of MCPyV have been reported in extracutaneous sites including the breast, salivary glands, oral mucosa, vaginal walls, lymph nodes, and esophagus [7,11]. The origin of MCC in extracutaneous sites including the stomach is difficult to explain. In these sites, neoplastic cells can arise either from Merkel cells migrating to the mucosa or, alternatively, from a stem cell that is able to differentiate assuming a Merkel cell phenotype. An origin from Merkel cells present in the stomach should be supported, considering that Merkel cells have been detected singly and in clusters in the human esophagus [12]. However, at present, no similar systematic studies have been conducted to prove the existence of Merkel cells in normal human gastric mucosa. An epidermal stem cell derivation, as an alternative to Merkel cell origin, has been considered for cutaneous MCCs to explain the presence of nonendocrine components sometimes observed in these carcinomas [13]. In the present gastric MCC, an origin from noncommitted progenitor cells that follow a pure Merkel cell differentiation may be hypothesized.

Primary gastric MCC harboring DNA polyomavirus An interesting finding of our case is the presence of MCPyV in tumor cells, demonstrated using PCR analysis, immunohistochemistry, and electron microscopy. This result confirms previous findings proving that MCPyV is present in extracutaneous MCCs including lymph nodes and parotid glands [7] and supports the concept that MCCs are multiorgan carcinomas. There is a fairly great morphologic and immunophenotypical overlap between MCC and PD-NECs arising in sites such as the lung (small cell carcinoma) and digestive tract. Consequently, visceral NECs have always to be considered in the differential diagnosis of MCC. MCCs show a characteristic finely dispersed chromatin of nuclei and immunoreactivity for cytokeratin 20 with a typical “dotlike” paranuclear signal, whereas they are negative for TTF1 [14]. The demonstration of the presence of MCPyV using the above-mentioned methods further supports the diagnosis of MCC because MCPyV has never been detected in visceral PD-NECs and in pulmonary neuroendocrine tumors [15]. In summary, we report and illustrate an unusual gastric MCC. This is a rare but distinct morphologic entity that can arise diagnostic challenges.

References [1] Kohler S, Kerl H. Merkel cell carcinoma. In: LeBoit PE, Burg G, Weedon D, Sarasin A, editors. World Health Organization classification of tumours. Pathology & genetics of skin tumours. Lyon, France: IARC Press; 2006. p. 272-3. [2] Canales LI, Parker A, Kadakia S. Upper gastrointestinal bleeding from Merkel cell carcinoma. Am J Gastroenterol 1992;87:1464-6. [3] Cubiella J, Salgado M, Riu M, et al. Gastric metastasis due to Merkel cell carcinoma: a rare cause of gastric bleeding. Rev Esp Enferm Dig 2004;96:150-1.

5 [4] Wolov K, Tully O, Mercogliano G. Gastric metastasis of Merkel cell carcinoma. Clin Gastroenterol Hepatol 2009;7:A26. [5] Rosa F, Pacelli F, Papa V, et al. Merkel cell carcinoma metastatic to the stomach. ANZ J Surg 2010;80:100-1. [6] Syal NG, Dang S, Rose J, Chen C, Aduli F. Gastric metastasis of Merkel cell cancer—uncommon complication of a rare neoplasm. J Ark Med Soc 2012;109:134-6. [7] de Biase D, Ragazzi M, Asioli S, Eusebi V. Extracutaneous Merkel cell carcinomas harbor polyomavirus DNA. HUM PATHOL 2012;43: 980-5. [8] Schowalter RM, Pastrana DV, Pumphrey KA, Moyer AL, Buck CB. Merkel cell polyomavirus and two previously unknown polyomaviruses are chronically shed from human skin. Cell Host Microbe 2010;7:509-15. [9] Tibiletti MG, Martin V, Bernasconi B, et al. BCL2, BCL6, MYC, MALT 1, and BCL10 rearrangements in nodal diffuse large B-cell lymphomas: a multicentre evaluation of a new set of fluorescent in situ hybridization probes and correlation with clinical outcome. HUM PATHOL 2009;40:645-52. [10] Connelly TJ, Cribier B, Brown TJ, Yanguas I. Complete spontaneous regression of Merkel cell carcinoma: a review of the 10 reported cases. Dermatol Surg 2000;26:853-6. [11] Albores-Saavedra J, Batich K, Chable-Montero F, Sagy N, Schwartz AM, Henson DE. Merkel cell carcinoma demographics, morphology, and survival based on 3870 cases: a population based study. J Cutan Pathol 2010;37:20-7. [12] Harmse JL, Carey FA, Baird AR, et al. Merkel cells in the human oesophagus. J Pathol 1999;189:176-9. [13] Walsh NM. Primary neuroendocrine (Merkel cell) carcinoma of the skin: morphologic diversity and implications thereof. HUM PATHOL 2001;32:680-9. [14] La Rosa S, Chiaravalli AM, Placidi C, Papanikolaou N, Cerati M, Capella C. TTF1 expression in normal lung neuroendocrine cells and related tumors. Immunohistochemical study comparing two different monoclonal antibodies. Virchows Arch 2010;457: 497-507. [15] Ly TY, Walsh NM, Pasternak S. The spectrum of Merkel cell polyomavirus expression in Merkel cell carcinoma, in a variety of cutaneous neoplasms, and in neuroendocrine carcinomas from different anatomical site. HUM PATHOL 2012;43:557-66.