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An update on diagnostic features of Merkel cell carcinoma
MINI-SYMPOSIUM: DERMATOPATHOLOGY
An update on diagnostic features of Merkel cell carcinoma
that expresses oncogenic viral proteins such as large T antigen (LT) and small T antigen (ST).7e10 MCC can hence be divided into two molecular subclasses: virus-positive MCC (VP-MCC) and virus-negative MCC (VN-MCC).2 Growing evidence suggests that differences between these subclasses have diagnostic and prognostic significance.11,12
Carole Bitar
Epidemiology
Douglas R Fullen
In 2013, the incidence rate of MCC in the United States was estimated at 0.7 cases/100,000 person-years, and is expected to steadily increase due to the aging population.13 MCC is 25 times more common in whites than other ethnic groups.1 There is an overall male predominance that may be accentuated among patients with VN-MCC.3,14 The median age at diagnosis is 76 years, and the majority of patients are older than 50.3 However, MCC has been reported in children and young adults, and can be more aggressive in this age group.15 Patients with immunosuppression, including human immunodeficiency virus, organ transplant and chronic lymphocytic leukemia, are at increased risk of developing MCC.1,2 MCC patients are at higher risk of developing second malignancies, mainly melanoma and hematologic malignancies, which affect overall survival.4
Paul W Harms
Abstract Merkel cell carcinoma (MCC) is an aggressive primary cutaneous neuroendocrine carcinoma that predominantly affects the elderly and immunosuppressed. Although rare, the incidence of MCC is increasing. Evidence suggests that the pathogenesis of MCC is related to Merkel cell polyomavirus infection or ultraviolet mutagenesis. Clinically, MCC typically presents as an asymptomatic violaceous papule on the head and neck. Histologically MCC is a small round blue cell tumor that must be differentiated from other small cell tumors, therefore immunohistochemistry is necessary for diagnosis of MCC. However, recent evidence suggests that some standard diagnostic markers may be less reliable in virus-negative MCC. MCC may develop local and distant metastases, with poor prognosis for advanced disease. Immunotherapy has recently been shown to be effective for many advanced cases. Here, we review diagnostic features of MCC including potential diagnostic and prognostic differences between virus-positive and virus-negative tumors.
Clinical presentation Clinical presentation of MCC can be variable and non-specific.1 The most common presentation is an erythematous painless nodule on the head and neck of elderly white men (Figure 1).1 These clinical features are summarized under the AEIOU acronym (asymptomatic/lack of tenderness, expanding rapidly, immune suppression, older than 50 years, ultraviolet-exposed site on fair skin).1,3 About two thirds of patients with MCC will have localized disease at presentation.1
Keywords cytokeratin-20; immunotherapy; Merkel cell carcinoma; Merkel cell polyomavirus; TTF-1
Introduction
Diagnostic features
Merkel cell carcinoma (MCC) is a rare, highly aggressive primary cutaneous neuroendocrine carcinoma.1e3 It most frequently affects elderly and immunosuppressed patients, and can be twice as lethal as melanoma.4 MCC was initially described in 1972 by Toker, who referred to it as trabecular carcinoma.5 Further studies demonstrated the presence of neurosecretory granules in these tumors, resembling those in Merkel cell mechanoreceptor cells.6 Evidence suggests that MCC may arise from two independent mechanisms: ultravioletinduced mutagenesis, or Merkel cell polyomavirus (MCPyV)
Scanning magnification At low power, MCC are blue cell tumors typically located in the dermis, with frequent involvement of the subcutis (Figure 2A). Epidermal involvement is very uncommon.3 Some tumors may infiltrate deeper tissue such as skeletal muscle. Rare intraepidermal cases (MCC in situ) have been reported.16 The growth pattern of MCC can be nodular (Figure 2A), infiltrative (Figure 2B), or a mixture of these patterns.3 Tumor cells may be arranged in a sheet-like or trabecular pattern (Figure 2C).3 Possible stromal changes include brisk inflammatory response, increased vascularity, mucin (Figure 2E) or amyloid.3,17 High magnification MCC is classically composed of monotonous basophilic cells with high nuclear to cytoplasmic ratio and a salt-and-pepper neuroendocrine chromatin (Figure 2D).3,17 The cells range in size from small (Figure 2E), intermediate, to large, with intermediate being the most common type.17 MCC cells have a hyperchromatic nucleus surrounded by a small rim of cytoplasm.3 Rarely, tumor cells have visible amphophilic cytoplasm with a relatively lower nuclear to cytoplasmic ratio and occasional pleomorphic nuclei (Figure 2F). High rates of mitosis and necrosis, as well as molding and crush artifact, are usually present.3 MCC commonly
Carole Bitar MD House Officer, Pathology, Department of Dermatology, Tulane University, New Orleans, LA, USA. Conflicts of interest: The author received research support from Celgene to conduct an investigator-initiated trial. There was no funding for this study. Douglas R. Fullen MD Professor, Departments of Pathology and Dermatology, University of Michigan, Ann Arbor, MI, USA. Conflicts of interest: The author reports no conflicts of interest. Paul W. Harms MD PhD Assistant Professor, Departments of Pathology and Dermatology, University of Michigan, Ann Arbor, MI, USA. Conflicts of interest: The author reports no conflicts of interest.
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neuroendocrine marker is completely sensitive for MCC, therefore maximal sensitivity is achieved by the use of multiple markers. Thyroid transcription factor 1 (TTF-1) is classically negative in MCC (Table 1, Figure 3E), which can differentiate MCC from metastatic SCLC.26,31,32 Recently, it has been reported that approximately a third of VN-MCC express TTF-1, although expression in such cases is focal and weak.12 MCC with squamous differentiation (combined tumors) have been reported to have distinctive immunophenotypic profiles from pure MCC tumors,12,20,21 although some differences may be related to virus-negative status rather than divergent differentiation.12 Squamous areas generally display a similar immunophenotypic profile to squamous cell carcinoma. Investigational markers reported to have high sensitivity for MCC include INSM1 and ATOH1 (Table 1).30,33,34
Figure 1 Clinical appearance of MCC. Erythematous nodule on the extremity of an elderly Caucasian individual. Tumors may also present on the head and neck, or less frequently the trunk.
Prognostic markers A number of prognostic markers have been proposed for MCC, although currently none have entered routine clinical use, and studies in the setting of immunotherapy have been limited.2 Certain inflammatory cell populations in the tumor microenvironment, including CD8 þ T-cells and CD33þ myeloid cells, have been associated with more favorable outcome.35,36 PD-L1 expression (based upon density or percentage) may correlate with response to immunotherapy.37,38 A recent large study found that MCC with MCPyV positivity carries a better prognosis than tumors with MCPyV negativity, although previous smaller studies had mixed results.11,39 In patients with VP-MCC tumors, a high titer of MCPyV viral capsid protein-1 serum antibodies or the presence of anti-ST serum antibodies at diagnosis is associated with a better outcome, whereas persistent or re-emergent anti-ST antibodies is a poor prognostic finding.40,41 Biomarkers reported to be associated with less favorable outcome include p63, vascular endothelial growth factor, CD34, EZH2, increased ATOH1, and nuclear expression of survivin.42e47
displays lymphovascular invasion.3,17 When present, intraepidermal involvement presents as pagetoid spread, either as cells with recognizably neuroendocrine chromatin, or as clusters of small hyperchromatic cells (Figure 2G, 2H) that may raise the differential diagnosis of squamous cell carcinoma in situ, melanoma in situ, or even mycosis fungoides.1,3,18,19 Heterogeneous differentiation toward squamous, eccrine, neuroblastic, melanocytic, and sarcomatoid morphologies has been described in MCC (Figure 2I, J).3 Of these, squamous differentiation is the most common, and appears to be specific to VN-MCC relative to VPMCC.12,20,21 MCC can also coexist with other malignancies, especially squamous cell carcinoma in situ (Figure 2H). Basal cell carcinoma, trichoblastoma, poroma, and cysts have been reported to occur alongside MCC.3,17
Ancillary studies Diagnostic immunohistochemistry MCC must be differentiated from metastatic small cell carcinoma (SmCC), especially small cell lung cancer (SCLC), and other poorly differentiated carcinomas.3,22 Immunohistochemical stains are required to confirm the diagnosis.3 MCC stains with a broad spectrum cytokeratin (CAM5.2, AE1/AE3, and 34bE12) with almost 100% sensitivity (Table 1).23,24 The cytokeratin staining patterns of MCC may be dot-like perinuclear, cytoplasmic, or mixed.23 Cytokeratin 20 (CK20), the classic marker for MCC, is highly sensitive and relatively specific, and displays staining patterns similar to other cytokeratins (Table 1, Figure 3A, 3B).25e27 CK20 expression is present in about 95% of MCC; in cases without CK20 expression, an expanded panel of immunohistochemical stains is necessary for diagnostic confirmation.3 CK7 is negative in most VP-MCC, but can be expressed in up to 46% of VN-MCC (Table 1).12 Neurofilament (NF) also displays dot-like perinuclear expression in MCC (Table 1, Figure 3C).23,28 NF is more specific than CK20 for MCC relative to other small cell carcinomas,29 and is frequently expressed in CK20-negative cases.29,30 NF has lower sensitivity in VN-MCC (50e75%) than VP-MCC (75e100%).12,29 MCC stains with neuroendocrine markers including chromogranin A, synaptophysin (Figure 3D), and neuron specific enolase (NSE), as well as the neural marker CD56.3,17 No single
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Merkel cell polyomavirus detection Merkel cell polyomavirus is a common virus in the healthy population.2 In rare circumstances MCPyV can play an oncogenic role, in which setting it is associated with MCC pathogenesis in about 80% of cases.2,10,11 Common methods for detection of MCPyV include immunohistochemistry using antibodies against the viral T antigen, and polymerase chain reaction (PCR) to detect viral DNA. The most widely reported antibody for immunohistochemistry is the commercially available antibody clone CM2B4, which is directed against LT (Figure 3G).48 Other antibodies, including the Ab3 antibody targeting LT, have also been investigated.49 Challenges with immunohistochemistry include the potential for equivocal or false negative results, and background nonspecific staining of lymphocytes in some cases.11,50 Quantitative PCR directed against viral LT and ST DNA sequences is sensitive for detecting the presence of MCPyV.49 However, low nonspecific signal (likely related to wild-type infectious virus in background skin) can complicate interpretation, especially in samples with lower tumor purity,14,51 and an optimal threshold for positivity has not been determined. Reports have been mixed regarding the relative sensitivity and specificity of these assays for MCPyV detection, as well as the
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Figure 2 Microscopic features of MCC. (a) Scanning magnification displaying nodular growth pattern with sheet-like growth of tumor cells. (b) Infiltrative growth pattern. (c) Trabecular arrangement. (d) Classic cytomorphology displaying small round blue cells with neuroendocrine chromatin, scant cytoplasm, and frequent mitoses. (e) MCC with small cell morphology and stromal mucin. (f) Rare cases demonstrate cells with characteristic neuroendocrine chromatin that have increased cytoplasm and occasional pleomorphic nuclei. (g) Pagetoid scatter of intraepidermal MCC. (h) Squamous cell carcinoma in situ associated with MCC. (i) Squamous differentiation, characterized by balls of squamous cells scattered throughout the MCC tumor. (j) Sarcomatoid change with neuroendocrine cells segueing into spindled cells. Hematoxylin and eosin stain, magnification 6x (a), 100x (b), 200x (c, h), 400x (d-g, i, j).
implication of virus positivity for clinical outcomes.11,39 The largest study, conducted by Moshiri et al., compared the sensitivity and specificity of quantitative PCR and immunohistochemistry (using antibodies Ab3 and CM2B4) to a multimodal approach integrating results from all three assays.11 By this
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approach, they found that immunohistochemistry by CM2B4 had 88% sensitivity and 94% specificity in detecting MCPyV, compared to PCR sensitivity of 82.5% and specificity of 81.1%.11 Immunohistochemistry using antibody Ab3 had the highest sensitivity (98.3%) but lowest specificity (45.3%). Other studies
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Immunohistochemical findings in MCC Marker
Basic diagnostic panel CK20
Pan-keratin
Expression
Relationship to MCPyV status
Comments
Positive (>90%), perinuclear dot and/or cytoplasmic
May be decreased in VN-MCC
Specific for MCC relative to other cutaneous carcinomas; infrequent expression in SCLC (14%); frequent expression in SmCC from certain other sites
May be reduced chromogranin A in VN-MCC
No single neuroendocrine marker is completely sensitive; also expressed in other carcinomas, including BCC Positive in SCLC
Neuroendocrine markers
Positive (w100%), perinuclear dot and/or cytoplasmic Positive
TTF-1
Negative (majority)
Expanded panel Neurofilament
Positive, perinuclear dot and/or cytoplasmic MCPyV Positive (60e80%), nuclear cytoplasmic CK7 Positive (minority), perinuclear dot and/or cytoplasmic Other markers (investigational or not specific to MCC) Melanocytic markers Negative SATB2 Positive ASCL1 (MASH1) Negative TdT Positive (minority) CD3, CD20, CD45 Negative PAX5 Positive (minority) BerEp4 Positive CD99 Positive (membranous or dot-like) p63 Positive (minority, weak) INSM1 Positive ATOH1 (HATH1) Positive BCL2 Positive (minority) EMA Positive
More frequent expression in VN-MCC Less frequent expression in VN-MCC Definitional
Often more subtle expression than CK20; rarely expressed in extracutaneous SmCC Rare to absent in non-MCC tumors
More frequent expression in VN-MCC
Positive in SCLC
Some reports of S100 expression in MCC Negative in SCLC Positive in SCLC Negative in SCLC Allow distinction from lymphoma Overlap with B-cell lymphoma
May be highly sensitive for MCC Also expressed in extracutaneous SmCC Also expressed in BCC
BCC: basal cell carcinoma. MCC: Merkel cell carcinoma. MCPyV: Merkel cell polyomavirus. SCLC: small cell lung carcinoma. SmCC: small cell carcinoma. VN-MCC: virusnegative Merkel cell carcinoma.
Table 1
on smaller cohorts have found PCR to be more sensitive than immunohistochemistry,48e50,52 and PCR sensitivity may be further improved by the use of additional primer sets.49 Additional approaches to MCPyV detection are under investigation. RNA in situ hybridization using probes that target LT and ST is highly sensitive for detecting MCPyV, and allows for correlation with tissue morphology (Figure 3G).14,19 DNA in situ hybridization has also been described.53 Next generation sequencing (NGS) approaches may provide the highest specificity in excluding background wild-type virus, by evaluating for tumor-specific MCPyV mutations, viral integration sites, and the presence or absence of mutations in the cellular genome; however, the optimal NGS approach has not yet been determined, and limitations include high cost and required expertise.2
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Differential diagnosis The differential diagnosis of MCC is extensive and includes basal cell carcinoma (BCC) or other cutaneous carcinomas, lymphoma, Ewing sarcoma (ES), melanoma, and metastatic SCLC.3,54 Careful interpretation of microscopic and immunohistochemical findings is necessary to distinguish these entities. Basal cell carcinoma BCC is the most common carcinoma in humans, and has characteristic peripheral palisading, retraction artifact, and stromal changes.55 In contrast to the elongated basaloid nuclei of BCC, MCC is composed of small round cells and has crush artifact not seen usually in BCC.17 Angiolymphatic invasion is frequently seen in MCC but rare in BCC.17 MCC and BCC can both demonstrate mucin and amyloid deposition, and peripheral
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Figure 3 Immunophenotypic findings of MCC. (a) CK20, perinuclear dot pattern. (b) CK20, mixed cytoplasmic and perinuclear patterns. (c) Neurofilament, perinuclear dot pattern. (d) Synaptophysin. (e) Absence of TTF1 staining. (f) LTAg IHC in MCPyV-positive MCC. (g) MCPyV RNAISH in MCPyV-positive MCC. Magnification: 400x (aef), 600x (g). DIAGNOSTIC HISTOPATHOLOGY 25:3
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clefting.17,25,56 Unlike BCC, clefting in MCC is not associated with peripheral palisading. Individual MCC tumor cells disperse into stromal mucin in a manner not seen in BCC (Figure 2E). Pagetoid scatter is observed in a minority of MCC, but is absent in BCC. In rare cases where these two entities cannot be distinguished by hematoxylin and eosin stains, immunohistochemical stains can be used to make the diagnosis. CK20 stains MCC in a dot-like pattern but is usually negative in BCC.23 MCPyV stain is highly specific for MCC and rarely is positive in BCC.57 Epithelial membrane antigen is positive in MCC and negative in BCC.24,58 Ber-EP4, BCL2, and neuroendocrine markers can be expressed in both tumor types (Table 1).17
and NF.23,25e27 Most MCC has a distinctive staining pattern (CK20þ, NFþ, MCPyVþ and TTF-1 negative), and the opposite pattern is classically seen in metastatic noncutaneous small cell carcinoma.7,12 However, CK20 is not completely specific for MCC, as it is frequently positive in SmCC arising in the salivary glands, uterine cervix, and genitourinary tract, and is focally expressed in 14% of SCLC.25,29,67 Diffuse CK20 expression is relatively more specific to MCC.29 However, a minority of MCC may have focal or absent CK20 expression, especially VN-MCC tumors.12,68 TTF-1 is expressed in a majority of SCLC22 and is rarely expressed in VP-MCC, but has been reported to be expressed in approximately one third of VN-MCC.12 TTF-1 is weak and focal when expressed in MCC,12 hence diffuse TTF-1 expression favors SCLC.22 NF and MCPyV have higher specificity than CK20 for distinguishing MCC from SCLC, although sensitivity is lower.3,29 MASH1 (ASCL1) is a marker for SCLC (positive in about 85%) that is negative in MCC.69 Because no marker is completely sensitive and specific for distinguishing MCC from SCLC, a panel of immunohistochemical stains, coupled with correlation with clinical history and any appropriate imaging, can be essential for excluding a non-cutaneous origin.
Other cutaneous carcinomas MCC may be misdiagnosed or overlooked in the presence of concurrent squamous cell carcinoma.3,59 Furthermore, there is potential for confusion between intraepidermal MCC and SCCIS in limited samplings. Cutaneous carcinomas that display a small blue cell pattern need to be distinguished from MCC. Small cell sweat gland carcinoma of childhood can look histologically identical to MCC, however it lacks the characteristic CK20 positivity of MCC, affects a younger population, and has a better prognosis.3 Nipple neuroendocrine carcinoma is another rare entity that mimics MCC but lacks CK20 positivity.17
Staging and prognosis MCC is an aggressive cutaneous neuroendocrine tumor with poor outcome at advanced stage.1,2 Most commonly MCC presents at a localized stage; however, regional metastases (typically in regional lymph nodes) and distant metastases are frequent, occurring in 26% and 8% of cases, respectively.70 MCC may also develop satellite and in transit metastases in regional skin. Distant sites of metastasis may include liver, bone, lung, and brain. The five year overall survival of MCC patients ranges from 14 to 51% depending on the stage at diagnosis.3 The clinical size of the tumor upon initial diagnosis is the most important prognostic factor; however, even small tumors can develop nodal metastases in up to 10e20% of cases.71 Patients with pathological confirmation of negative lymph node disease have a better prognosis than patients with clinically negative lymph node disease.70 Thus, sentinel lymph node biopsy has been recommended for all stages of MCC.1 Clinically occult nodal disease carries a better prognosis than clinically positive nodal disease, with the exception of nodal metastases of unknown primary.70 An increasing number of lymph nodes involved by metastasis, and higher tumor burden within the lymph node, predict a worse prognosis.72,73 Common patterns of lymph node involvement by MCC include subcapsular sinusoidal, parafollicular, sheet-like, or rare scattered parenchymal cells; of these, sheet-like involvement may portend the worst prognosis.72 However, as metastases of any size and pattern are currently considered positive for staging, immunohistochemistry with pankeratin and CK 20 is recommended to identify nodal metastases.29,74 When evaluating lymph nodes for metastatic MCC, the potential for lymph node involvement by chronic lymphocytic leukemia (which may have been previously undiagnosed) must be kept in mind.2 In a small percentage of cases, a patient with MCC may develop a second, clonally unrelated MCC tumor.70,75 As histopathologic criteria for distinguishing primary MCC from cutaneous metastases have not been established, this distinction is
Melanoma Both melanoma and MCC may demonstrate pagetoid spread and intraepidermal nesting.17 Melanocytic markers including HMB45 and S100 are usually negative in MCC, although S100 staining has been reported for some cases of MCC.60 NSE, a marker of MCC, can stain melanoma as well.60 Thus, a panel of melanocytic and epithelial markers is useful when melanoma enters the differential diagnosis.60 Lymphoma MCC can express lymphocytic markers including terminal deoxynucleotidyl transferase (TdT), PAX5, and immunoglobulins (Table 1).61e64 This staining pattern, together with the propensity for MCC to arise in the setting of B-cell lymphoma, can make distinction between MCC and lymphoma challenging. In rare cases, MCC can co-occur with cutaneous T-cell lymphoma,19 in which setting intraepidermal MCC may be confused with Pautrier microabscesses.18,19 However, lymphoma has characteristic markers that are negative in MCC including CD45, CD3 and CD20 (Table 1).61 Ewing sarcoma family tumors Primary cutaneous ES is a rare small blue cell tumor that expresses CD99 and Fli-1.17 This immunohistochemical pattern is not unique for primary cutaneous ES, as MCC can stain with CD99 (membranous or dot pattern) and Fli-1.17,24,65 Both MCC and ES stain with NSE.17,24 CK20 expression has not been reported in ES.65 Testing for EWSR1 rearrangement can be useful in challenging cases, as this rearrangement has not been identified in MCC.66 Metastatic noncutaneous small cell carcinoma Metastatic noncutaneous SmCC such as SCLC can be histologically indistinguishable from MCC. Immunohistochemical markers used to distinguish the two include CK20, MCPyV, TTF-1
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currently made by clinical correlation. Epidermotropism may be observed in both primary and metastatic tumors.3 Although the presence of concurrent squamous cell carcinoma has been proposed as evidence for primary disease, clinically metastatic MCC with a concurrent squamous cell carcinoma has been described.59 Molecular studies may be helpful in distinction of a new primary tumor from a metastasis.59,75e78
Immunotherapy for treatment of MCC Avelumab is a PDL-1 inhibitor that was FDA-approved in 2018 for treatment of metastatic MCC.2,92 Other anti-PD1 and PDL-1 inhibitors have also shown promise.93e95 Another potential immune-based strategy is reversal of major histocompatibility complex 1 (MHC1) down-regulation on tumor cells, to promote tumor antigenicity.96 Interleukin-12, ipilimumab and toll-like receptor 4 agonists are being studied in clinical trials for the treatment of advanced MCC.4 Adoptive transfer of polyomavirusspecific T cells in conjunction with other therapies was effective in one case.97
Treatment MCC is an aggressive tumor that requires a multidisciplinary approach for optimal management. Localized MCC disease is treated with wide local surgical excision with 1e2 cm margins.2 Sentinel lymph node biopsy is recommended for all patients with MCC, even for small tumors.1 Adjuvant radiotherapy may be used at the primary site, and at the nodal basin following the identification of metastatic disease; the most effective use for radiotherapy is under investigation.2 Chemotherapy might be used in advanced stage IV disease as a palliative treatment without a durable response.2 Immunotherapy is now the leading treatment modality for patients with advanced disease.2
Summary MCC is an aggressive cutaneous neuroendocrine tumor with high morbidity and mortality. MCPyV and UV-induced mutagenesis play fundamental roles in the pathogenesis of VP-MCC and VNMCC, respectively. Recent molecular analyses of MCC revealed a potential for targeted therapy and immunotherapy. MCC must be distinguished from other small blue cell tumors using immunohistochemical markers. Wide local excision and radiotherapy are the recommended therapy for localized disease. Avelumab, a PDL-1 inhibitor, was recently approved for metastatic MCC with more durable response than chemotherapy. Further studies are needed to define predictive markers for immunotherapy response, and to identify alternative therapeutic options for nonresponders. Recent evidence indicates that VPMCC and VN-MCC tumors may differ in diagnostic marker expression and clinical course. A
Molecular features of MCC MCC pathogenesis MCC pathogenesis arises in association with one of two proposed etiologies: MCPyV and photodamage.7e10 Although MCPyV is frequently detectable in the skin of healthy individuals, the rate of malignant transformation is exceptionally low.2 Two events are required for oncogenic transformation: integration of the polyomavirus genome into the host genome, and truncation of LT to render the virus replication-deficient.2 The truncated LT antigen inactivates the tumor suppressor retinoblastoma (Rb).2 ST has complex activities modulating protein stability, chromatin remodeling, mRNA translation, and nuclear factor-kB signaling.2,79 VN-MCC is associated with a substantial burden of UV-signature mutations and highly recurrent inactivation of tumor suppressor genes TP53 and RB1.7e9,80
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Activation of oncogenes in MCC Oncogenes are activated in a minority of MCC and represent potential therapeutic targets. Phosphoinositide 3-kinase pathway activation in MCC occurs by activating mutations of AKT1 and PIK3CA genes, and is more frequently observed in VN-MCC.81,82 Phosphoinositide 3-kinase inhibitors have been studied in vivo and in vitro on MCC with promising results.81e83 Receptor tyrosine kinase activating events are not prevalent in MCC, limiting the potential for tyrosine kinase inhibitors as targeted therapy.2 MYCL1 (L-Myc), which has been shown to represent a therapeutic target via BET bromodomain inhibitors in other SmCC,84 is amplified in about 39% of MCC.85 The oncogene EZH2 is overexpressed in a subset of MCC and might be a target for treatment.46,86,87 Preclinical data suggest Bcl-2 and survivin as potential targets in MCC.88e90 Due to the heterogeneity of oncogene activation events in MCC, tumor mutation profiling may be necessary to identify potential therapeutic susceptibilities for any given MCC tumor.91
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10 Feng H, Shuda M, Chang Y, Moore PS. Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science 2008; 319: 1096e100. 11 Moshiri AS, Doumani R, Yelistratova L, et al. Polyomavirusnegative Merkel cell carcinoma: a more aggressive subtype based on analysis of 282 cases using multimodal tumor virus detection. J Invest Dermatol 2017; 137: 819e27. 12 Pasternak S, Carter MD, Ly TY, Doucette S, Walsh NM. Immunohistochemical profiles of different subsets of Merkel cell carcinoma. Hum Pathol 2018; 82. 13 Paulson KG, Park SY, Vandeven NA, et al. Merkel cell carcinoma: current US incidence and projected increases based on changing demographics. J Am Acad Dermatol 2018; 78: 457e63. e452. 14 Wang L, Harms PW, Palanisamy N, et al. Age and gender associations of virus positivity in Merkel cell carcinoma characterized using a novel RNA in situ hybridization assay. Clin Canc Res 2017; 23: 5622e30. 15 Paulson KG, Nghiem P. One in a hundred million: Merkel cell carcinoma in pediatric and young adult patients is rare but more likely to present at advanced stages based on US registry data. J Am Acad Dermatol 2018; 80. 16 Jour G, Aung PP, Rozas-Munoz E, Curry JL, Prieto V, Ivan D. Intraepidermal Merkel cell carcinoma: a case series of a rare entity with clinical follow up. J Cutan Pathol 2017; 44: 684e91. 17 Pulitzer MP, Amin BD, Busam KJ. Merkel cell carcinoma: review. Adv Anat Pathol 2009; 16: 135e44. 18 Rocamora A, Badia N, Vives R, Carrillo R, Ulloa J, Ledo A. Epidermotropic primary neuroendocrine (Merkel cell) carcinoma of the skin with Pautrier-like microabscesses. Report of three cases and review of the literature. J Am Acad Dermatol 1987; 16: 1163e8. 19 Zoumberos N, McMullen E, Wang L, et al. Merkel cell carcinoma arising in association with cutaneous T-cell lymphoma: a potential diagnostic pitfall. J Cutan Pathol, 2018; https://doi.org/10.1111/ cup.13404 [Epub ahead of print]. 20 Pulitzer MP, Brannon AR, Berger MF, et al. Cutaneous squamous and neuroendocrine carcinoma: genetically and immunohistochemically different from Merkel cell carcinoma. Mod Pathol 2015; 28: 1023e32. 21 Busam KJ, Jungbluth AA, Rekthman N, et al. Merkel cell polyomavirus expression in Merkel cell carcinomas and its absence in combined tumors and pulmonary neuroendocrine carcinomas. Am J Surg Pathol 2009; 33: 1378e85. 22 Hanly AJ, Elgart GW, Jorda M, Smith J, Nadji M. Analysis of thyroid transcription factor-1 and cytokeratin 20 separates Merkel cell carcinoma from small cell carcinoma of lung. J Cutan Pathol 2000; 27: 118e20. 23 Schmidt U, Muller U, Metz KA, Leder LD. Cytokeratin and neurofilament protein staining in Merkel cell carcinoma of the small cell type and small cell carcinoma of the lung. Am J Dermatopathol 1998; 20: 346e51. 24 Llombart B, Monteagudo C, Lopez-Guerrero JA, et al. Clinicopathological and immunohistochemical analysis of 20 cases of Merkel cell carcinoma in search of prognostic markers. Histopathology 2005; 46: 622e34. 25 Chan JK, Suster S, Wenig BM, Tsang WY, Chan JB, Lau AL. Cytokeratin 20 immunoreactivity distinguishes Merkel cell (primary cutaneous neuroendocrine) carcinomas and salivary gland
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small cell carcinomas from small cell carcinomas of various sites. Am J Surg Pathol 1997; 21: 226e34. Cheuk W, Kwan MY, Suster S, Chan JK. Immunostaining for thyroid transcription factor 1 and cytokeratin 20 aids the distinction of small cell carcinoma from Merkel cell carcinoma, but not pulmonary from extrapulmonary small cell carcinomas. Arch Pathol Lab Med 2001; 125: 228e31. Bobos M, Hytiroglou P, Kostopoulos I, Karkavelas G, Papadimitriou CS. Immunohistochemical distinction between Merkel cell carcinoma and small cell carcinoma of the lung. Am J Dermatopathol 2006; 28: 99e104. McCalmont TH. Paranuclear dots of neurofilament reliably identify Merkel cell carcinoma. J Cutan Pathol 2010; 37: 821e3. Stanoszek LM, Chan MP, Palanisamy N, et al. Neurofilament is superior to cytokeratin 20 in supporting cutaneous origin for neuroendocrine carcinoma. Histopathology, 2018; https://doi.org/ 10.1111/his.13758 [Epub ahead of print]. Kervarrec T, Tallet A, Miquelestorena-Standley E, et al. Diagnostic accuracy of a panel of immunohistochemical and molecular markers to distinguish Merkel cell carcinoma from other neuroendocrine carcinomas. Mod Pathol, 2018; https://doi.org/10. 1038/s41379-018-0155-y [Epub ahead of print]. Byrd-Gloster AL, Khoor A, Glass LF, et al. Differential expression of thyroid transcription factor 1 in small cell lung carcinoma and Merkel cell tumor. Hum Pathol 2000; 31: 58e62. Ordonez NG. Value of thyroid transcription factor-1 immunostaining in distinguishing small cell lung carcinomas from other small cell carcinomas. Am J Surg Pathol 2000; 24: 1217e23. Rush PS, Rosenbaum JN, Roy M, Baus RM, Bennett DD, Lloyd RV. Insulinoma-associated 1: a novel nuclear marker in Merkel cell carcinoma (cutaneous neuroendocrine carcinoma). J Cutan Pathol 2018; 45: 129e35. Leonard JH, Cook AL, Van Gele M, et al. Proneural and proneuroendocrine transcription factor expression in cutaneous mechanoreceptor (Merkel) cells and Merkel cell carcinoma. Int J Canc 2002; 101: 103e10. Paulson KG, Iyer JG, Tegeder AR, et al. Transcriptome-wide studies of Merkel cell carcinoma and validation of intratumoral CD8þ lymphocyte invasion as an independent predictor of survival. J Clin Oncol 2011; 29: 1539e46. Kervarrec T, Gaboriaud P, Berthon P, et al. Merkel cell carcinomas infiltrated with CD33(þ) myeloid cells and CD8(þ) T cells are associated with improved outcome. J Am Acad Dermatol 2018; 78: 973e82. e978. Giraldo NA, Nguyen P, Engle EL, et al. Multidimensional, quantitative assessment of PD-1/PD-L1 expression in patients with Merkel cell carcinoma and association with response to pembrolizumab. J Immunother Cancer 2018; 6: 99. Kaufman HL, Russell JS, Hamid O, et al. Updated efficacy of avelumab in patients with previously treated metastatic Merkel cell carcinoma after >/¼1 year of follow-up: JAVELIN Merkel 200, a phase 2 clinical trial. J Immunother Cancer 2018; 6: 7. Coursaget P, Samimi M, Nicol JT, Gardair C, Touze A. Human Merkel cell polyomavirus: virological background and clinical implications. Apmis 2013; 121: 755e69. Touze A, Le Bidre E, Laude H, et al. High levels of antibodies against Merkel cell polyomavirus identify a subset of patients with
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Merkel cell carcinoma with better clinical outcome. J Clin Oncol 2011; 29: 1612e9. Paulson KG, Carter JJ, Johnson LG, et al. Antibodies to Merkel cell polyomavirus T antigen oncoproteins reflect tumor burden in Merkel cell carcinoma patients. Cancer Res 2010; 70: 8388e97. Asioli S, Righi A, Volante M, Eusebi V, Bussolati G. p63 expression as a new prognostic marker in Merkel cell carcinoma. Cancer 2007; 110: 640e7. Ng L, Beer TW, Murray K. Vascular density has prognostic value in Merkel cell carcinoma. Am J Dermatopathol 2008; 30: 442e5. Kim J, McNiff JM. Nuclear expression of survivin portends a poor prognosis in Merkel cell carcinoma. Mod Pathol 2008; 21: 764e9. Fernandez-Figueras MT, Puig L, Musulen E, et al. Expression profiles associated with aggressive behavior in Merkel cell carcinoma. Mod Pathol 2007; 20: 90e101. Harms KL, Chubb H, Zhao L, et al. Increased expression of EZH2 in Merkel cell carcinoma is associated with disease progression and poorer prognosis. Hum Pathol 2017; 67: 78e84. Gambichler T, Mohtezebsade S, Wieland U, et al. Prognostic relevance of high atonal homolog-1 expression in Merkel cell carcinoma. J Canc Res Clin Oncol 2017; 143: 43e9. Shuda M, Arora R, Kwun HJ, et al. Human Merkel cell polyomavirus infection I. MCV T antigen expression in Merkel cell carcinoma, lymphoid tissues and lymphoid tumors. Int J Canc 2009; 125: 1243e9. Rodig SJ, Cheng J, Wardzala J, et al. Improved detection suggests all Merkel cell carcinomas harbor Merkel polyomavirus. J Clin Invest 2012; 122: 4645e53. Leroux-Kozal V, Leveque N, Brodard V, et al. Merkel cell carcinoma: histopathologic and prognostic features according to the immunohistochemical expression of Merkel cell polyomavirus large T antigen correlated with viral load. Hum Pathol 2015; 46: 443e53. Eid M, Nguyen J, Brownell I. Seeking standards for the detection of Merkel cell polyomavirus and its clinical significance. J Invest Dermatol 2017; 137: 797e9. Shuda M, Kwun HJ, Feng H, Chang Y, Moore PS. Human Merkel cell polyomavirus small T antigen is an oncoprotein targeting the 4E-BP1 translation regulator. J Clin Invest 2011; 121: 3623e34. Haugg AM, Rennspiess D, zur Hausen A, et al. Fluorescence in situ hybridization and qPCR to detect Merkel cell polyomavirus physical status and load in Merkel cell carcinomas. Int J Canc 2014; 135: 2804e15. Succaria F, Radfar A, Bhawan J. Merkel cell carcinoma (primary neuroendocrine carcinoma of skin) mimicking basal cell carcinoma with review of different histopathologic features. Am J Dermatopathol 2014; 36: 160e6. Paolino G, Donati M, Didona D, Mercuri SR, Cantisani C. Histology of non-melanoma skin cancers: an update. Biomedicines 2017; 5. Plaza JA, Suster S. The Toker tumor: spectrum of morphologic features in primary neuroendocrine carcinomas of the skin (Merkel cell carcinoma). Ann Diagn Pathol 2006; 10: 376e85. Mertz KD, Paasinen A, Arnold A, et al. Merkel cell polyomavirus large T antigen is detected in rare cases of nonmelanoma skin cancer. J Cutan Pathol 2013; 40: 543e9. Heyderman E, Graham RM, Chapman DV, Richardson TC, McKee PH. Epithelial markers in primary skin cancer: an immunoperoxidase study of the distribution of epithelial membrane
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antigen (EMA) and carcinoembryonic antigen (CEA) in 65 primary skin carcinomas. Histopathology 1984; 8: 423e34. Falto Aizpurua LA, Wang M, Ruiz HA, et al. A case of combined Merkel cell carcinoma and squamous cell carcinoma: molecular insights and diagnostic pitfalls. JAAD Case Rep 2018; 4: 996e9. Kontochristopoulos GJ, Stavropoulos PG, Krasagakis K, Goerdt S, Zouboulis CC. Differentiation between Merkel cell carcinoma and malignant melanoma: an immunohistochemical study. Dermatology 2000; 201: 123e6. Sur M, AlArdati H, Ross C, Alowami S. TdT expression in Merkel cell carcinoma: potential diagnostic pitfall with blastic hematological malignancies and expanded immunohistochemical analysis. Mod Pathol 2007; 20: 1113e20. Dong HY, Liu W, Cohen P, Mahle CE, Zhang W. B-cell specific activation protein encoded by the PAX-5 gene is commonly expressed in Merkel cell carcinoma and small cell carcinomas. Am J Surg Pathol 2005; 29: 687e92. Buresh CJ, Oliai BR, Miller RT. Reactivity with TdT in Merkel cell carcinoma: a potential diagnostic pitfall. Am J Clin Pathol 2008; 129: 894e8. Kolhe R, Reid MD, Lee JR, Cohen C, Ramalingam P. Immunohistochemical expression of PAX5 and TdT by Merkel cell carcinoma and pulmonary small cell carcinoma: a potential diagnostic pitfall but useful discriminatory marker. Int J Clin Exp Pathol 2013; 6: 142e7. Nicholson SA, McDermott MB, Swanson PE, Wick MR. CD99 and cytokeratin-20 in small-cell and basaloid tumors of the skin. Appl Immunohistochem Mol Morphol 2000; 8: 37e41. Fernandez-Flores A, Suarez-Penaranda JM, Alonso S. Study of EWS/FLI-1 rearrangement in 18 cases of CK20þ/CM2B4þ Merkel cell carcinoma using FISH and correlation to the differential diagnosis of Ewing sarcoma/peripheral neuroectodermal tumor. Appl Immunohistochem Mol Morphol 2013; 21: 379e85. McCluggage WG, Kennedy K, Busam KJ. An immunohistochemical study of cervical neuroendocrine carcinomas: neoplasms that are commonly TTF1 positive and which may express CK20 and P63. Am J Surg Pathol 2010; 34: 525e32. Miner AG, Patel RM, Wilson DA, et al. Cytokeratin 20-negative Merkel cell carcinoma is infrequently associated with the Merkel cell polyomavirus. Mod Pathol 2015; 28: 498e504. Ralston J, Chiriboga L, Nonaka D. MASH1: a useful marker in differentiating pulmonary small cell carcinoma from Merkel cell carcinoma. Mod Pathol 2008; 21: 1357e62. Harms KL, Healy MA, Nghiem P, et al. Analysis of prognostic factors from 9387 Merkel cell carcinoma cases forms the basis for the new 8th edition AJCC staging system. Ann Surg Oncol 2016; 23: 3564e71. Schwartz JL, Griffith KA, Lowe L, et al. Features predicting sentinel lymph node positivity in Merkel cell carcinoma. J Clin Oncol 2011; 29: 1036e41. Ko JS, Prieto VG, Elson PJ, et al. Histological pattern of Merkel cell carcinoma sentinel lymph node metastasis improves stratification of Stage III patients. Mod Pathol 2016; 29: 122e30. Iyer JG, Storer BE, Paulson KG, et al. Relationships among primary tumor size, number of involved nodes, and survival for 8044 cases of Merkel cell carcinoma. J Am Acad Dermatol 2014; 70: 637e43.
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86 Veija T, Koljonen V, Bohling T, Kero M, Knuutila S, Sarhadi VK. Aberrant expression of ALK and EZH2 in Merkel cell carcinoma. BMC Canc 2017; 17: 236. 87 Harms PW, Collie AM, Hovelson DH, et al. Next generation sequencing of Cytokeratin 20-negative Merkel cell carcinoma reveals ultraviolet-signature mutations and recurrent TP53 and RB1 inactivation. Mod Pathol 2016; 29: 240e8. 88 Verhaegen ME, Mangelberger D, Weick JW, et al. Merkel cell carcinoma dependence on bcl-2 family members for survival. J Invest Dermatol 2014; 134: 2241e50. 89 Dresang LR, Guastafierro A, Arora R, Normolle D, Chang Y, Moore PS. Response of Merkel cell polyomavirus-positive Merkel cell carcinoma xenografts to a survivin inhibitor. PLoS One 2013; 8: e80543. 90 Arora R, Shuda M, Guastafierro A, et al. Survivin is a therapeutic target in Merkel cell carcinoma. Sci Transl Med 2012; 4: 133ra156. 91 Cassler NM, Merrill D, Bichakjian CK, Brownell I. Merkel cell carcinoma therapeutic update. Curr Treat Options Oncol 2016; 17: 36. 92 Kaufman HL, Russell J, Hamid O, et al. Avelumab in patients with chemotherapy-refractory metastatic Merkel cell carcinoma: a multicentre, single-group, open-label, phase 2 trial. Lancet Oncol 2016; 17: 1374e85. 93 Walocko FM, Scheier BY, Harms PW, Fecher LA, Lao CD. Metastatic Merkel cell carcinoma response to nivolumab. J Immunother Cancer 2016; 4: 79. 94 Nghiem PT, Bhatia S, Lipson EJ, et al. PD-1 blockade with pembrolizumab in advanced merkel-cell carcinoma. N Engl J Med 2016; 374: 2542e52. 95 Baker M, Cordes L, Brownell I. Avelumab: a new standard for treating metastatic Merkel cell carcinoma. Expert Rev Anticancer Ther 2018; 18: 319e26. 96 Paulson KG, Tegeder A, Willmes C, et al. Downregulation of MHCI expression is prevalent but reversible in Merkel cell carcinoma. Cancer Immunol Res 2014; 2: 1071e9. 97 Chapuis AG, Afanasiev OK, Iyer JG, et al. Regression of metastatic Merkel cell carcinoma following transfer of polyomavirusspecific T cells and therapies capable of re-inducing HLA class-I. Cancer Immunol Res 2014; 2: 27e36.
74 Su LD, Lowe L, Bradford CR, Yahanda AI, Johnson TM, Sondak VK. Immunostaining for cytokeratin 20 improves detection of micrometastatic Merkel cell carcinoma in sentinel lymph nodes. J Am Acad Dermatol 2002; 46: 661e6. 75 Harms KL, Lazo de la Vega L, Hovelson DH, et al. Molecular profiling of multiple primary Merkel cell carcinoma to distinguish genetically distinct tumors from clonally related metastases. JAMA Dermatol 2017; 153: 505e12. 76 Eluri M, Feneran A, Bordeaux JS, et al. Multiple Merkel cell carcinomas: late metastasis or multiple primary tumors? A molecular study. JAAD Case Rep 2017; 3: 131e4. 77 Ahronowitz IZ, Daud AI, Leong SP, et al. An isolated Merkel cell carcinoma metastasis at a distant cutaneous site presenting as a second ’primary’ tumor. J Cutan Pathol 2011; 38: 801e7. 78 Schrama D, Thiemann A, Houben R, Kahler KC, Becker JC, Hauschild A. Distinction of 2 different primary Merkel cell carcinomas in 1 patient by Merkel cell polyomavirus genome analysis. Arch Dermatol 2010; 146: 687e9. 79 Kwun HJ, Shuda M, Feng H, Camacho CJ, Moore PS, Chang Y. Merkel cell polyomavirus small T antigen controls viral replication and oncoprotein expression by targeting the cellular ubiquitin ligase SCFFbw7. Cell Host Microbe 2013; 14: 125e35. 80 Cimino PJ, Robirds DH, Tripp SR, Pfeifer JD, Abel HJ, Duncavage EJ. Retinoblastoma gene mutations detected by whole exome sequencing of Merkel cell carcinoma. Mod Pathol 2014; 27: 1073e87. 81 Hafner C, Houben R, Baeurle A, et al. Activation of the PI3K/AKT pathway in Merkel cell carcinoma. PLoS One 2012; 7. e31255. 82 Nardi V, Song Y, Santamaria-Barria JA, et al. Activation of PI3K signaling in Merkel cell carcinoma. Clin Canc Res 2012; 18: 1227e36. 83 Shiver MB, Mahmoud F, Gao L. Response to idelalisib in a patient with stage IV merkel-cell carcinoma. N Engl J Med 2015; 373: 1580e2. 84 Kato F, Fiorentino FP, Alibes A, et al. MYCL is a target of a BET bromodomain inhibitor, JQ1, on growth suppression efficacy in small cell lung cancer cells. Oncotarget 2016; 7: 77378e88. 85 Paulson KG, Lemos BD, Feng B, et al. Array-CGH reveals recurrent genomic changes in Merkel cell carcinoma including amplification of L-Myc. J Invest Dermatol 2009; 129: 1547e55.
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An update on diagnostic features of Merkel cell carcinoma
that expresses oncogenic viral proteins such as large T antigen (LT) and small T antigen (ST).7e10 MCC can hence be divided into two molecular subclasses: virus-positive MCC (VP-MCC) and virus-negative MCC (VN-MCC).2 Growing evidence suggests that differences between these subclasses have diagnostic and prognostic significance.11,12
Carole Bitar
Epidemiology
Douglas R Fullen
In 2013, the incidence rate of MCC in the United States was estimated at 0.7 cases/100,000 person-years, and is expected to steadily increase due to the aging population.13 MCC is 25 times more common in whites than other ethnic groups.1 There is an overall male predominance that may be accentuated among patients with VN-MCC.3,14 The median age at diagnosis is 76 years, and the majority of patients are older than 50.3 However, MCC has been reported in children and young adults, and can be more aggressive in this age group.15 Patients with immunosuppression, including human immunodeficiency virus, organ transplant and chronic lymphocytic leukemia, are at increased risk of developing MCC.1,2 MCC patients are at higher risk of developing second malignancies, mainly melanoma and hematologic malignancies, which affect overall survival.4
Paul W Harms
Abstract Merkel cell carcinoma (MCC) is an aggressive primary cutaneous neuroendocrine carcinoma that predominantly affects the elderly and immunosuppressed. Although rare, the incidence of MCC is increasing. Evidence suggests that the pathogenesis of MCC is related to Merkel cell polyomavirus infection or ultraviolet mutagenesis. Clinically, MCC typically presents as an asymptomatic violaceous papule on the head and neck. Histologically MCC is a small round blue cell tumor that must be differentiated from other small cell tumors, therefore immunohistochemistry is necessary for diagnosis of MCC. However, recent evidence suggests that some standard diagnostic markers may be less reliable in virus-negative MCC. MCC may develop local and distant metastases, with poor prognosis for advanced disease. Immunotherapy has recently been shown to be effective for many advanced cases. Here, we review diagnostic features of MCC including potential diagnostic and prognostic differences between virus-positive and virus-negative tumors.
Clinical presentation Clinical presentation of MCC can be variable and non-specific.1 The most common presentation is an erythematous painless nodule on the head and neck of elderly white men (Figure 1).1 These clinical features are summarized under the AEIOU acronym (asymptomatic/lack of tenderness, expanding rapidly, immune suppression, older than 50 years, ultraviolet-exposed site on fair skin).1,3 About two thirds of patients with MCC will have localized disease at presentation.1
Keywords cytokeratin-20; immunotherapy; Merkel cell carcinoma; Merkel cell polyomavirus; TTF-1
Introduction
Diagnostic features
Merkel cell carcinoma (MCC) is a rare, highly aggressive primary cutaneous neuroendocrine carcinoma.1e3 It most frequently affects elderly and immunosuppressed patients, and can be twice as lethal as melanoma.4 MCC was initially described in 1972 by Toker, who referred to it as trabecular carcinoma.5 Further studies demonstrated the presence of neurosecretory granules in these tumors, resembling those in Merkel cell mechanoreceptor cells.6 Evidence suggests that MCC may arise from two independent mechanisms: ultravioletinduced mutagenesis, or Merkel cell polyomavirus (MCPyV)
Scanning magnification At low power, MCC are blue cell tumors typically located in the dermis, with frequent involvement of the subcutis (Figure 2A). Epidermal involvement is very uncommon.3 Some tumors may infiltrate deeper tissue such as skeletal muscle. Rare intraepidermal cases (MCC in situ) have been reported.16 The growth pattern of MCC can be nodular (Figure 2A), infiltrative (Figure 2B), or a mixture of these patterns.3 Tumor cells may be arranged in a sheet-like or trabecular pattern (Figure 2C).3 Possible stromal changes include brisk inflammatory response, increased vascularity, mucin (Figure 2E) or amyloid.3,17 High magnification MCC is classically composed of monotonous basophilic cells with high nuclear to cytoplasmic ratio and a salt-and-pepper neuroendocrine chromatin (Figure 2D).3,17 The cells range in size from small (Figure 2E), intermediate, to large, with intermediate being the most common type.17 MCC cells have a hyperchromatic nucleus surrounded by a small rim of cytoplasm.3 Rarely, tumor cells have visible amphophilic cytoplasm with a relatively lower nuclear to cytoplasmic ratio and occasional pleomorphic nuclei (Figure 2F). High rates of mitosis and necrosis, as well as molding and crush artifact, are usually present.3 MCC commonly
Carole Bitar MD House Officer, Pathology, Department of Dermatology, Tulane University, New Orleans, LA, USA. Conflicts of interest: The author received research support from Celgene to conduct an investigator-initiated trial. There was no funding for this study. Douglas R. Fullen MD Professor, Departments of Pathology and Dermatology, University of Michigan, Ann Arbor, MI, USA. Conflicts of interest: The author reports no conflicts of interest. Paul W. Harms MD PhD Assistant Professor, Departments of Pathology and Dermatology, University of Michigan, Ann Arbor, MI, USA. Conflicts of interest: The author reports no conflicts of interest.
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neuroendocrine marker is completely sensitive for MCC, therefore maximal sensitivity is achieved by the use of multiple markers. Thyroid transcription factor 1 (TTF-1) is classically negative in MCC (Table 1, Figure 3E), which can differentiate MCC from metastatic SCLC.26,31,32 Recently, it has been reported that approximately a third of VN-MCC express TTF-1, although expression in such cases is focal and weak.12 MCC with squamous differentiation (combined tumors) have been reported to have distinctive immunophenotypic profiles from pure MCC tumors,12,20,21 although some differences may be related to virus-negative status rather than divergent differentiation.12 Squamous areas generally display a similar immunophenotypic profile to squamous cell carcinoma. Investigational markers reported to have high sensitivity for MCC include INSM1 and ATOH1 (Table 1).30,33,34
Figure 1 Clinical appearance of MCC. Erythematous nodule on the extremity of an elderly Caucasian individual. Tumors may also present on the head and neck, or less frequently the trunk.
Prognostic markers A number of prognostic markers have been proposed for MCC, although currently none have entered routine clinical use, and studies in the setting of immunotherapy have been limited.2 Certain inflammatory cell populations in the tumor microenvironment, including CD8 þ T-cells and CD33þ myeloid cells, have been associated with more favorable outcome.35,36 PD-L1 expression (based upon density or percentage) may correlate with response to immunotherapy.37,38 A recent large study found that MCC with MCPyV positivity carries a better prognosis than tumors with MCPyV negativity, although previous smaller studies had mixed results.11,39 In patients with VP-MCC tumors, a high titer of MCPyV viral capsid protein-1 serum antibodies or the presence of anti-ST serum antibodies at diagnosis is associated with a better outcome, whereas persistent or re-emergent anti-ST antibodies is a poor prognostic finding.40,41 Biomarkers reported to be associated with less favorable outcome include p63, vascular endothelial growth factor, CD34, EZH2, increased ATOH1, and nuclear expression of survivin.42e47
displays lymphovascular invasion.3,17 When present, intraepidermal involvement presents as pagetoid spread, either as cells with recognizably neuroendocrine chromatin, or as clusters of small hyperchromatic cells (Figure 2G, 2H) that may raise the differential diagnosis of squamous cell carcinoma in situ, melanoma in situ, or even mycosis fungoides.1,3,18,19 Heterogeneous differentiation toward squamous, eccrine, neuroblastic, melanocytic, and sarcomatoid morphologies has been described in MCC (Figure 2I, J).3 Of these, squamous differentiation is the most common, and appears to be specific to VN-MCC relative to VPMCC.12,20,21 MCC can also coexist with other malignancies, especially squamous cell carcinoma in situ (Figure 2H). Basal cell carcinoma, trichoblastoma, poroma, and cysts have been reported to occur alongside MCC.3,17
Ancillary studies Diagnostic immunohistochemistry MCC must be differentiated from metastatic small cell carcinoma (SmCC), especially small cell lung cancer (SCLC), and other poorly differentiated carcinomas.3,22 Immunohistochemical stains are required to confirm the diagnosis.3 MCC stains with a broad spectrum cytokeratin (CAM5.2, AE1/AE3, and 34bE12) with almost 100% sensitivity (Table 1).23,24 The cytokeratin staining patterns of MCC may be dot-like perinuclear, cytoplasmic, or mixed.23 Cytokeratin 20 (CK20), the classic marker for MCC, is highly sensitive and relatively specific, and displays staining patterns similar to other cytokeratins (Table 1, Figure 3A, 3B).25e27 CK20 expression is present in about 95% of MCC; in cases without CK20 expression, an expanded panel of immunohistochemical stains is necessary for diagnostic confirmation.3 CK7 is negative in most VP-MCC, but can be expressed in up to 46% of VN-MCC (Table 1).12 Neurofilament (NF) also displays dot-like perinuclear expression in MCC (Table 1, Figure 3C).23,28 NF is more specific than CK20 for MCC relative to other small cell carcinomas,29 and is frequently expressed in CK20-negative cases.29,30 NF has lower sensitivity in VN-MCC (50e75%) than VP-MCC (75e100%).12,29 MCC stains with neuroendocrine markers including chromogranin A, synaptophysin (Figure 3D), and neuron specific enolase (NSE), as well as the neural marker CD56.3,17 No single
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Merkel cell polyomavirus detection Merkel cell polyomavirus is a common virus in the healthy population.2 In rare circumstances MCPyV can play an oncogenic role, in which setting it is associated with MCC pathogenesis in about 80% of cases.2,10,11 Common methods for detection of MCPyV include immunohistochemistry using antibodies against the viral T antigen, and polymerase chain reaction (PCR) to detect viral DNA. The most widely reported antibody for immunohistochemistry is the commercially available antibody clone CM2B4, which is directed against LT (Figure 3G).48 Other antibodies, including the Ab3 antibody targeting LT, have also been investigated.49 Challenges with immunohistochemistry include the potential for equivocal or false negative results, and background nonspecific staining of lymphocytes in some cases.11,50 Quantitative PCR directed against viral LT and ST DNA sequences is sensitive for detecting the presence of MCPyV.49 However, low nonspecific signal (likely related to wild-type infectious virus in background skin) can complicate interpretation, especially in samples with lower tumor purity,14,51 and an optimal threshold for positivity has not been determined. Reports have been mixed regarding the relative sensitivity and specificity of these assays for MCPyV detection, as well as the
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Figure 2 Microscopic features of MCC. (a) Scanning magnification displaying nodular growth pattern with sheet-like growth of tumor cells. (b) Infiltrative growth pattern. (c) Trabecular arrangement. (d) Classic cytomorphology displaying small round blue cells with neuroendocrine chromatin, scant cytoplasm, and frequent mitoses. (e) MCC with small cell morphology and stromal mucin. (f) Rare cases demonstrate cells with characteristic neuroendocrine chromatin that have increased cytoplasm and occasional pleomorphic nuclei. (g) Pagetoid scatter of intraepidermal MCC. (h) Squamous cell carcinoma in situ associated with MCC. (i) Squamous differentiation, characterized by balls of squamous cells scattered throughout the MCC tumor. (j) Sarcomatoid change with neuroendocrine cells segueing into spindled cells. Hematoxylin and eosin stain, magnification 6x (a), 100x (b), 200x (c, h), 400x (d-g, i, j).
implication of virus positivity for clinical outcomes.11,39 The largest study, conducted by Moshiri et al., compared the sensitivity and specificity of quantitative PCR and immunohistochemistry (using antibodies Ab3 and CM2B4) to a multimodal approach integrating results from all three assays.11 By this
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approach, they found that immunohistochemistry by CM2B4 had 88% sensitivity and 94% specificity in detecting MCPyV, compared to PCR sensitivity of 82.5% and specificity of 81.1%.11 Immunohistochemistry using antibody Ab3 had the highest sensitivity (98.3%) but lowest specificity (45.3%). Other studies
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Immunohistochemical findings in MCC Marker
Basic diagnostic panel CK20
Pan-keratin
Expression
Relationship to MCPyV status
Comments
Positive (>90%), perinuclear dot and/or cytoplasmic
May be decreased in VN-MCC
Specific for MCC relative to other cutaneous carcinomas; infrequent expression in SCLC (14%); frequent expression in SmCC from certain other sites
May be reduced chromogranin A in VN-MCC
No single neuroendocrine marker is completely sensitive; also expressed in other carcinomas, including BCC Positive in SCLC
Neuroendocrine markers
Positive (w100%), perinuclear dot and/or cytoplasmic Positive
TTF-1
Negative (majority)
Expanded panel Neurofilament
Positive, perinuclear dot and/or cytoplasmic MCPyV Positive (60e80%), nuclear cytoplasmic CK7 Positive (minority), perinuclear dot and/or cytoplasmic Other markers (investigational or not specific to MCC) Melanocytic markers Negative SATB2 Positive ASCL1 (MASH1) Negative TdT Positive (minority) CD3, CD20, CD45 Negative PAX5 Positive (minority) BerEp4 Positive CD99 Positive (membranous or dot-like) p63 Positive (minority, weak) INSM1 Positive ATOH1 (HATH1) Positive BCL2 Positive (minority) EMA Positive
More frequent expression in VN-MCC Less frequent expression in VN-MCC Definitional
Often more subtle expression than CK20; rarely expressed in extracutaneous SmCC Rare to absent in non-MCC tumors
More frequent expression in VN-MCC
Positive in SCLC
Some reports of S100 expression in MCC Negative in SCLC Positive in SCLC Negative in SCLC Allow distinction from lymphoma Overlap with B-cell lymphoma
May be highly sensitive for MCC Also expressed in extracutaneous SmCC Also expressed in BCC
BCC: basal cell carcinoma. MCC: Merkel cell carcinoma. MCPyV: Merkel cell polyomavirus. SCLC: small cell lung carcinoma. SmCC: small cell carcinoma. VN-MCC: virusnegative Merkel cell carcinoma.
Table 1
on smaller cohorts have found PCR to be more sensitive than immunohistochemistry,48e50,52 and PCR sensitivity may be further improved by the use of additional primer sets.49 Additional approaches to MCPyV detection are under investigation. RNA in situ hybridization using probes that target LT and ST is highly sensitive for detecting MCPyV, and allows for correlation with tissue morphology (Figure 3G).14,19 DNA in situ hybridization has also been described.53 Next generation sequencing (NGS) approaches may provide the highest specificity in excluding background wild-type virus, by evaluating for tumor-specific MCPyV mutations, viral integration sites, and the presence or absence of mutations in the cellular genome; however, the optimal NGS approach has not yet been determined, and limitations include high cost and required expertise.2
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Differential diagnosis The differential diagnosis of MCC is extensive and includes basal cell carcinoma (BCC) or other cutaneous carcinomas, lymphoma, Ewing sarcoma (ES), melanoma, and metastatic SCLC.3,54 Careful interpretation of microscopic and immunohistochemical findings is necessary to distinguish these entities. Basal cell carcinoma BCC is the most common carcinoma in humans, and has characteristic peripheral palisading, retraction artifact, and stromal changes.55 In contrast to the elongated basaloid nuclei of BCC, MCC is composed of small round cells and has crush artifact not seen usually in BCC.17 Angiolymphatic invasion is frequently seen in MCC but rare in BCC.17 MCC and BCC can both demonstrate mucin and amyloid deposition, and peripheral
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Figure 3 Immunophenotypic findings of MCC. (a) CK20, perinuclear dot pattern. (b) CK20, mixed cytoplasmic and perinuclear patterns. (c) Neurofilament, perinuclear dot pattern. (d) Synaptophysin. (e) Absence of TTF1 staining. (f) LTAg IHC in MCPyV-positive MCC. (g) MCPyV RNAISH in MCPyV-positive MCC. Magnification: 400x (aef), 600x (g). DIAGNOSTIC HISTOPATHOLOGY 25:3
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clefting.17,25,56 Unlike BCC, clefting in MCC is not associated with peripheral palisading. Individual MCC tumor cells disperse into stromal mucin in a manner not seen in BCC (Figure 2E). Pagetoid scatter is observed in a minority of MCC, but is absent in BCC. In rare cases where these two entities cannot be distinguished by hematoxylin and eosin stains, immunohistochemical stains can be used to make the diagnosis. CK20 stains MCC in a dot-like pattern but is usually negative in BCC.23 MCPyV stain is highly specific for MCC and rarely is positive in BCC.57 Epithelial membrane antigen is positive in MCC and negative in BCC.24,58 Ber-EP4, BCL2, and neuroendocrine markers can be expressed in both tumor types (Table 1).17
and NF.23,25e27 Most MCC has a distinctive staining pattern (CK20þ, NFþ, MCPyVþ and TTF-1 negative), and the opposite pattern is classically seen in metastatic noncutaneous small cell carcinoma.7,12 However, CK20 is not completely specific for MCC, as it is frequently positive in SmCC arising in the salivary glands, uterine cervix, and genitourinary tract, and is focally expressed in 14% of SCLC.25,29,67 Diffuse CK20 expression is relatively more specific to MCC.29 However, a minority of MCC may have focal or absent CK20 expression, especially VN-MCC tumors.12,68 TTF-1 is expressed in a majority of SCLC22 and is rarely expressed in VP-MCC, but has been reported to be expressed in approximately one third of VN-MCC.12 TTF-1 is weak and focal when expressed in MCC,12 hence diffuse TTF-1 expression favors SCLC.22 NF and MCPyV have higher specificity than CK20 for distinguishing MCC from SCLC, although sensitivity is lower.3,29 MASH1 (ASCL1) is a marker for SCLC (positive in about 85%) that is negative in MCC.69 Because no marker is completely sensitive and specific for distinguishing MCC from SCLC, a panel of immunohistochemical stains, coupled with correlation with clinical history and any appropriate imaging, can be essential for excluding a non-cutaneous origin.
Other cutaneous carcinomas MCC may be misdiagnosed or overlooked in the presence of concurrent squamous cell carcinoma.3,59 Furthermore, there is potential for confusion between intraepidermal MCC and SCCIS in limited samplings. Cutaneous carcinomas that display a small blue cell pattern need to be distinguished from MCC. Small cell sweat gland carcinoma of childhood can look histologically identical to MCC, however it lacks the characteristic CK20 positivity of MCC, affects a younger population, and has a better prognosis.3 Nipple neuroendocrine carcinoma is another rare entity that mimics MCC but lacks CK20 positivity.17
Staging and prognosis MCC is an aggressive cutaneous neuroendocrine tumor with poor outcome at advanced stage.1,2 Most commonly MCC presents at a localized stage; however, regional metastases (typically in regional lymph nodes) and distant metastases are frequent, occurring in 26% and 8% of cases, respectively.70 MCC may also develop satellite and in transit metastases in regional skin. Distant sites of metastasis may include liver, bone, lung, and brain. The five year overall survival of MCC patients ranges from 14 to 51% depending on the stage at diagnosis.3 The clinical size of the tumor upon initial diagnosis is the most important prognostic factor; however, even small tumors can develop nodal metastases in up to 10e20% of cases.71 Patients with pathological confirmation of negative lymph node disease have a better prognosis than patients with clinically negative lymph node disease.70 Thus, sentinel lymph node biopsy has been recommended for all stages of MCC.1 Clinically occult nodal disease carries a better prognosis than clinically positive nodal disease, with the exception of nodal metastases of unknown primary.70 An increasing number of lymph nodes involved by metastasis, and higher tumor burden within the lymph node, predict a worse prognosis.72,73 Common patterns of lymph node involvement by MCC include subcapsular sinusoidal, parafollicular, sheet-like, or rare scattered parenchymal cells; of these, sheet-like involvement may portend the worst prognosis.72 However, as metastases of any size and pattern are currently considered positive for staging, immunohistochemistry with pankeratin and CK 20 is recommended to identify nodal metastases.29,74 When evaluating lymph nodes for metastatic MCC, the potential for lymph node involvement by chronic lymphocytic leukemia (which may have been previously undiagnosed) must be kept in mind.2 In a small percentage of cases, a patient with MCC may develop a second, clonally unrelated MCC tumor.70,75 As histopathologic criteria for distinguishing primary MCC from cutaneous metastases have not been established, this distinction is
Melanoma Both melanoma and MCC may demonstrate pagetoid spread and intraepidermal nesting.17 Melanocytic markers including HMB45 and S100 are usually negative in MCC, although S100 staining has been reported for some cases of MCC.60 NSE, a marker of MCC, can stain melanoma as well.60 Thus, a panel of melanocytic and epithelial markers is useful when melanoma enters the differential diagnosis.60 Lymphoma MCC can express lymphocytic markers including terminal deoxynucleotidyl transferase (TdT), PAX5, and immunoglobulins (Table 1).61e64 This staining pattern, together with the propensity for MCC to arise in the setting of B-cell lymphoma, can make distinction between MCC and lymphoma challenging. In rare cases, MCC can co-occur with cutaneous T-cell lymphoma,19 in which setting intraepidermal MCC may be confused with Pautrier microabscesses.18,19 However, lymphoma has characteristic markers that are negative in MCC including CD45, CD3 and CD20 (Table 1).61 Ewing sarcoma family tumors Primary cutaneous ES is a rare small blue cell tumor that expresses CD99 and Fli-1.17 This immunohistochemical pattern is not unique for primary cutaneous ES, as MCC can stain with CD99 (membranous or dot pattern) and Fli-1.17,24,65 Both MCC and ES stain with NSE.17,24 CK20 expression has not been reported in ES.65 Testing for EWSR1 rearrangement can be useful in challenging cases, as this rearrangement has not been identified in MCC.66 Metastatic noncutaneous small cell carcinoma Metastatic noncutaneous SmCC such as SCLC can be histologically indistinguishable from MCC. Immunohistochemical markers used to distinguish the two include CK20, MCPyV, TTF-1
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currently made by clinical correlation. Epidermotropism may be observed in both primary and metastatic tumors.3 Although the presence of concurrent squamous cell carcinoma has been proposed as evidence for primary disease, clinically metastatic MCC with a concurrent squamous cell carcinoma has been described.59 Molecular studies may be helpful in distinction of a new primary tumor from a metastasis.59,75e78
Immunotherapy for treatment of MCC Avelumab is a PDL-1 inhibitor that was FDA-approved in 2018 for treatment of metastatic MCC.2,92 Other anti-PD1 and PDL-1 inhibitors have also shown promise.93e95 Another potential immune-based strategy is reversal of major histocompatibility complex 1 (MHC1) down-regulation on tumor cells, to promote tumor antigenicity.96 Interleukin-12, ipilimumab and toll-like receptor 4 agonists are being studied in clinical trials for the treatment of advanced MCC.4 Adoptive transfer of polyomavirusspecific T cells in conjunction with other therapies was effective in one case.97
Treatment MCC is an aggressive tumor that requires a multidisciplinary approach for optimal management. Localized MCC disease is treated with wide local surgical excision with 1e2 cm margins.2 Sentinel lymph node biopsy is recommended for all patients with MCC, even for small tumors.1 Adjuvant radiotherapy may be used at the primary site, and at the nodal basin following the identification of metastatic disease; the most effective use for radiotherapy is under investigation.2 Chemotherapy might be used in advanced stage IV disease as a palliative treatment without a durable response.2 Immunotherapy is now the leading treatment modality for patients with advanced disease.2
Summary MCC is an aggressive cutaneous neuroendocrine tumor with high morbidity and mortality. MCPyV and UV-induced mutagenesis play fundamental roles in the pathogenesis of VP-MCC and VNMCC, respectively. Recent molecular analyses of MCC revealed a potential for targeted therapy and immunotherapy. MCC must be distinguished from other small blue cell tumors using immunohistochemical markers. Wide local excision and radiotherapy are the recommended therapy for localized disease. Avelumab, a PDL-1 inhibitor, was recently approved for metastatic MCC with more durable response than chemotherapy. Further studies are needed to define predictive markers for immunotherapy response, and to identify alternative therapeutic options for nonresponders. Recent evidence indicates that VPMCC and VN-MCC tumors may differ in diagnostic marker expression and clinical course. A
Molecular features of MCC MCC pathogenesis MCC pathogenesis arises in association with one of two proposed etiologies: MCPyV and photodamage.7e10 Although MCPyV is frequently detectable in the skin of healthy individuals, the rate of malignant transformation is exceptionally low.2 Two events are required for oncogenic transformation: integration of the polyomavirus genome into the host genome, and truncation of LT to render the virus replication-deficient.2 The truncated LT antigen inactivates the tumor suppressor retinoblastoma (Rb).2 ST has complex activities modulating protein stability, chromatin remodeling, mRNA translation, and nuclear factor-kB signaling.2,79 VN-MCC is associated with a substantial burden of UV-signature mutations and highly recurrent inactivation of tumor suppressor genes TP53 and RB1.7e9,80
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