biomedicines

Review of Non- : An Update

Giovanni Paolino 1,2, Michele Donati 3, Dario Didona 4 ID , Santo Raffaele Mercuri 1 and Carmen Cantisani 2,5,* ID

1 and Cosmetology, IRCCS San Raffaele Hospital, 20132 Milan, Italy; [email protected] (G.P.); [email protected] (S.R.M.) 2 Department of Dermatology, Dipartimento di Medicina Interna e Specialità Mediche, La Sapienza University of Rome, 00161 Rome, Italy 3 Department of , Università Campus Biomedico, 00161 Rome, Italy; [email protected] 4 First Division of Dermatology, IRCCS Istituto Dermopatico dell’Immacolata, 00161 Rome, Italy; [email protected] 5 Unità Operativa Complessa of Dermatology, Policlinico Umberto I, Sapienza University of Rome, Viale del Policlinico 155, 00161 Rome, Italy * Correspondence: [email protected] or [email protected]; Tel.: +39-064-997-6993; Fax: +39-0649-0243

Received: 9 November 2017; Accepted: 19 December 2017; Published: 20 December 2017

Abstract: Non-melanoma skin (NMSC) is the most frequently diagnosed cancer in humans. Several different non-melanoma skin cancers have been reported in the literature, with several histologic variants that frequently cause important differential diagnoses with other cutaneous tumors basal (BCC) is the most common malignant skin tumor, with different histologic variants that are associated with a greater or less aggressive behavior and that usually may be confused with other primitive skin tumors. Actinic , Bowen’s , , and invasive (SCC) correspond to the other line of NMSC, that may have only local tumoral behavior, easy to treat and with local management (as in the case of (AK), Bowen’s disease, and keratoacanthoma) or a more aggressive behavior with a potential metastatic spread, as in case of invasive SCC. Therefore, serves as the gold standard during daily clinical practice, in order to improve the therapeutical approaches to patients with NMSC and to understand the distinct histopathological features of NMSC. Here, we reported the main pathological features of different non-melanoma skin cancers.

Keywords: non-melanoma skin cancers; basal cell carcinoma; squamous cell carcinoma; diagnosis; pathology

1. Introduction is a worldwide, emerging clinical need in the elderly white population, with a steady increase in incidence rates, morbidity, and related medical costs [1]. Skin cancer is a heterogeneous group of cancers comprising cutaneous melanoma and non-melanoma skin cancers (NMSC), which predominantly affects patients older than 65 years of age [1]. Under the umbrella of NMSC are all the non-melanoma malignant affecting the skin [1]. However, especially epidemiologically, the term NMSC practically refers to , namely basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and actinic keratosis (AK). Because they account for the 99% of the tumors in this group, we will focus on these types of NMSC.

Biomedicines 2017, 5, 71; doi:10.3390/biomedicines5040071 www.mdpi.com/journal/biomedicines Biomedicines 2017, 5, 71 2 of 12

2. Discussion

2.1. Basal Cell Carcinoma and Its Histological Variants

2.1.1. General Considerations BCC is a cutaneous malignant proliferation, which derives from basaloid cells and accounts for 50% of all cancers in the United States [1–3]. It has been postulated that BCC derives from the basal cell layer and outer root sheath of the hair follicle; these cells are pluripotent epithelial cells [1]. In addition, the expression of CD10 emphasizes the follicular derivation of these epithelial cells; more specifically, the absence of cytokeratin 15 in BCC suggests that these pluripotent cells arise from the bulge region of the hair follicle [1,4]. Strong -exposure, , beta-Human (HPV), and human immunodeficiency virus (HIV) increase the risk of BCC in the general population [1].

2.1.2. Main Histologic Variants Several different variants of BCC have been reported, which show variable outcomes and prognoses [1]. In this paragraph, we analyze the different BCC histologic patterns, according to the different types that clinicians could find in the daily clinical practice (Table1).

Table 1. Main histological differential diagnoses of basal cell carcinoma (BCC).

Differential Diagnosis Pathological Features Tricoblastoma Absence of cleft, rudimentary hair germs, papillary mesenchymal bodies. Lack of basaloid cells disposed in peripheral palisades; adenoid-cystic ADC lesion without connection to the ; absence of artefactual clefts Bland , , ductal differentiation CEA+, EMA+ and MAC BerEp4- Rims of collagen bundles, calcification, follicular/sebaceous/infundibular differentiation and cut artefacts. Cytokeratin (CK)20+, p75+, Pleckstrin Tricoepithelioma * homology-like domain family A member 1 + (PHLDA1+), common acute lymphoblastic leukemiaantigen + (CD10+) in tumor stroma, CK 6-, Ki-67- and Androgen Rceptor- (AR-) Cells arranged in a diffuse, trabecular and/or nested pattern, involving also MCC the subcutis. Mouse Anti-Cytokeratin (CAM) 5.2+, CK20+, S100-, human leukocyte common antigen- ( LCA-), thyroid transcription factor 1- (TTF1-) ADC: ; MAC: microcystic adnexal carcinoma; MCC: Merkel cell carcinoma; * above all desmoplastic tricoepithelioma.

The nodular type accounts for 50% of all BCC [1] (Figures1 and2). Nodular BCC (NBCC) is characterized by aggregates of basaloid cells with well-defined borders, showing a peripheral palisading of cells and a typical cleft [1,5,6]. Central with eosinophilic, granular features may be also present, as well as mucin [1–3]. The heavy aggregates of mucin determine a cystic structure; calcification may be also present, especially in long-standing lesions [1]. Mitotic activity is usually not so evident, but a high mitotic rate may be present in more aggressive lesions [1]. Adenoidal BCC can be classified as a variant of NBCC, characterized by basaloid cells with a reticulated configuration extending into the [1–3,7,8]. The main differential diagnosis of adenoid BCC is adenoid cystic carcinoma and sometimes it is very difficult to differentiate the nature of the basaloid tumor cells, since in adenoid cystic carcinoma there are usually basaloid tumor cells with hyperchromatic nucleus arranged in a cribriform pattern undergoing dedifferentiation within a collagenous stroma. However, the lack of basalioid cells disposed in peripheral palisades in an adenoid cystic lesion without connection to the overlying epidermis and the absence of artefactual clefts between the tumor and the stroma help the distinction between an adenoid cystic carcinoma and BCC [2,3]. NBCC with granular features and large cytoplasmic inclusions has been defined as granular cell basal cell carcinoma, characterized by an eosinophilic and granular cytoplasm [1,9]. Interestingly, it has Biomedicines 2017, 5, 71 3 of 12 been reported that a percentage of BCC may show clear cells, which may be present at the periphery of the lesion, as well as in the center [1]. These cells are round or polyhedral with pale, eosinophilic, Biomedicines 2017, 5, 71 3 of 12 vacuolated,Biomedicines or finely 2017, 5 granular, 71 cytoplasm [9]. Basal cell epithelioma, another variant3 of of 12 NBCC, is periphery of the lesion, as well as in the center [1]. These cells are round or polyhedral with pale, characterizedperiphery by monster of the lesion, cells, as well which as in are the enlarged, center [1]. These mononuclear, cells are round and/or or polyhedral multinucleated with pale, [1,10,11]. eosinophilic, vacuolated, or finely granular cytoplasm [9]. Basal cell epithelioma, another variant of BCC witheosinophilic, outer hair vacuolated, follicle sheath or finely differentiation granular cytoplasm is another [9]. Basal variant,cell epithelioma, characterized another variant by infundibular of NBCC, is characterized by monster cells, which are enlarged, mononuclear, and/or multinucleated NBCC, is characterized by monster cells, which are enlarged, mononuclear, and/or multinucleated structures[1,10,11]. of the outer BCC with sheath outer [1 ].hair follicle sheath differentiation is another variant, characterized by [1,10,11]. BCC with outer hair follicle sheath differentiation is another variant, characterized by Micro-nodularinfundibular BCCstructures ischaracterized of the outer sheath by [1]. a plaque-like shape, showing an increase incidence infundibular structures of the outer sheath [1]. Micro-nodular BCC is characterized by a plaque-like shape, showing an increase incidence of of recurrenceMicro-nodular [1]. Pathologically, BCC is characterized it is similar by a to plaque-like NBCC, butshape, it showing is smaller an increase and forms incidence micro-nodules of recurrence [1]. Pathologically, it is similar to NBCC, but it is smaller and forms micro-nodules of of basaloidrecurrence cells,approximately [1]. Pathologically,the it is sizesimilar of to the NBCC, hair but bulbs, it is smaller with and minimal forms micro-nodules palisading of [1 ,12]. The basaloid cells, approximately the size of the hair bulbs, with minimal palisading [1,12]. The basaloid cells, approximately the size of the hair bulbs, with minimal palisading [1,12]. The surroundingsurrounding stroma isstroma myxoid is myxoid [1]. [1]. surrounding stroma is myxoid [1].

Figure 1. Nodular basal cell carcinoma (hematoxylin and eosin, 20×). FigureFigure 1. Nodular 1. Nodular basal basal cell cell carcinoma carcinoma (hematoxylin (hematoxylin and andeosin, eosin, 20×). 20×).

Figure 2. Nodular basal cell carcinoma (hematoxylin and eosin, 20×). FigureFigure 2. Nodular 2. Nodular basal basal cell cell carcinoma carcinoma (hematoxylin (hematoxylin and andeosin, eosin, 20×). 20×). Superficial BCC is another common variant of BCC [1]. It is characterized by nests of basaloid Superficial BCC is another common variant of BCC [1]. It is characterized by nests of basaloid cells that extend from the epidermis, with neoplastic cells that resemble primordial germ cells. Superficialcells that BCC extend is another from the common epidermis, variant with neoplastic of BCC [cells1]. It that is characterizedresemble primordial by nests germ of cells. basaloid cells Peripheral palisading is usually prominent and tumor islands show a well demarcated border [1]. Peripheral palisading is usually prominent and tumor islands show a well demarcated border [1]. that extend fromThe theinfiltrative epidermis, variant withof BCC neoplastic is a continuum cells between that resemble the nodular primordial and morpheiform germ cells.variant Peripheral The infiltrative variant of BCC is a continuum between the nodular and morpheiform variant palisading[11]. is usually This variant prominent is characterized and tumor by the islandspresenceshow of atypical a well basaloid demarcated cells in nodules border that [1]. show [11]. This variant is characterized by the presence of atypical basaloid cells in nodules that show The infiltrative variant of BCC is a continuum between the nodular and morpheiform variant [11]. This variant is characterized by the presence of atypical basaloid cells in nodules that show different sizes, with a surrounding mucinous stroma [1,13]. The of the sub-cutis and of the muscle, as well as of the adnexal structures, may be present. According to these features, this variant of BCC is more aggressive and more difficult to eradicate [1,13]. Another aggressive variant is morpheiphorm BCC, also known as fibrosing BCC [1,14]. This variant, also called sclerosing BCC, Biomedicines 2017, 5, 71 4 of 12

different sizes, with a surrounding mucinous stroma [1,13]. The invasion of the sub-cutis and of the muscle, as well as of the adnexal structures, may be present. According to these features, this variant of BCC is more aggressive and more difficult to eradicate [1,13]. Another aggressive variant is morpheiphorm BCC, also known as fibrosing BCC [1,14]. This variant, also called sclerosing BCC, is mainly characterized by the presence of thin and elongated tumor islands [14,15]. The peripheral palisading is absent and the stroma retraction is also infrequent [14,15]. The mitotic activity is usually detectable, although not so marked. Morpheiform BCC may involve the fat and cutaneous annexes [1,15]. Metatypical carcinoma (BCC with squamous differentiation) is a BCC with features of nodular BCC and SCC [12]. This variant is characterized by basaloid cells with variable eosinophilic features, Biomedicines 2017prominent, 5, 71 mitotic activity, and numerous apoptotic cells [16,17] (Figure 3). Peripheral palisading is 4 of 12 not always present. Metatypical carcinoma is one of the most aggressive subtypes, with a higher incidence of peri-neural and lymphatic spread [1,16,17]. Contrariwise, the term baso-squamous is mainly characterizedcarcinoma should by be theused presence in the case ofof tumors thin and with elongated contiguous tumorareas of islandsBCC and [SCC14,15 [1]]. (Figure The peripheral palisading4). is In absentthis case, andthe typical the stromafeatures of retraction both tumors is are also present infrequent [18]. [14,15]. The mitotic activity is The main histologic differential diagnoses of BCC are trichoblastoma, merkel cell carcinoma, usually detectable,desmoplastic although , not so marked.and microcystic Morpheiform adnexal carcinoma BCC [1] may (Table involve 1). the fat and cutaneous annexes [1,15].Specifically, trichlobastoma shows a peri-tumoral stroma that is dense and circumscribed, Metatypicalabsence of carcinoma clefting between (BCC tumor with cells squamous and stroma differentiation) (while it is present is in a BCCBCC), withrudimentary features hair of nodular BCC and SCCgerms [ 12with]. This epithelial variant strands is characterized enclosing primitive by basaloid mesenchyme, cells with also variable known eosinophilicas ‘papillary features, mesenchymal bodies’, features that are absent in BCC. Finally, the peripheral palisading is more prominentprominent mitotic activity, in BCC, although and numerous it can also apoptotic be found also cells in trichoblastoma [16,17] (Figure [2,3]3). (Table Peripheral 1). palisading is not always present.Merkel Metatypical cell carcinoma carcinoma is characterized is one of theby deeply most aggressivebasophilic tumoral subtypes, cells withwith asmall-to- higher incidence of peri-neuralmedium-sized and lymphatic cells, sparse spread cytoplasm, [1, 16with,17 dense]. Contrariwise, round nuclei (Figure the term5). Tumor baso-squamous cells are arranged carcinoma should bein used a diffuse, in the trabecular, case of tumors and/or nested with contiguouspattern, usually areas involving of BCC the and subcutis. SCC Contrariwise, [1] (Figure4 BCC). In this case, shows a more asymmetrical architecture, palisading of peripheral tumor cells, and a typical cleft the typicalbetween features tumor of bothcells and tumors stroma are (absent present in Merkel [18]. cell carcinoma) [2,3] (Table 1).

Figure 3. Metatypical basal cell carcinoma (hematoxylin and eosin, 20×). Figure 3. Metatypical basal cell carcinoma (hematoxylin and eosin, 20×). Biomedicines 2017, 5, 71 5 of 12

Figure 4. Basosquamous carcinoma (hematoxylin and eosin, 20×). Figure 4. Basosquamous carcinoma (hematoxylin and eosin, 20×).

The main histologic differential diagnoses of BCC are trichoblastoma, merkel cell carcinoma, desmoplastic trichoepithelioma, and microcystic adnexal carcinoma [1] (Table1). Specifically, trichlobastoma shows a peri-tumoral stroma that is dense and circumscribed, absence of clefting between tumor cells and stroma (while it is present in BCC), rudimentary hair germs with epithelial strands enclosing primitive mesenchyme, also known as ‘papillary mesenchymal bodies’, features that are absent in BCC. Finally, the peripheral palisading is more prominent in BCC, although it can also be found also in trichoblastoma [2,3] (Table1).

Figure 5. Typical small-to-medium-sized basophilic tumoral cells in a Merkel cell carcinoma. (hematoxilin and eosin, 30×).

Clinical and histopathological differentiation between morpheaform BCC and trichoepithelioma (above all desmoplastic tricoepithelioma) is usually a frequent problem (Figure 6). BCC is often an asymmetrical lesion, while trichoepithelioma is symmetrical, horn cysts are more frequent in trichoepithelioma, the ulceration is frequent in BCC and rare in trichoepithelioma [2,3]. Besides, rims of collagen bundles, calcification, follicular/sebaceous/infundibular differentiation, and cut artefacts are usually present in trichoepithelioma, while they are not present in BCC. Morpheaform BCC express usually epithelial cell adhesion molecule (EPCAM) (BerEP4), cytokeratin 6 (CK6, that is negative in tricoepithelioma), Ki-67 (negative in trichoepithelioma), and androgen receptor (AR) [19– 21]. Finally, CD10+ is positive in BCC tumor cells and positive in the stroma in tricoepithelioma. cytokeratin 20 (CK20) is present in tricoepithelioma, as well as p75 and PHLDA1 are positive mainly in desmoplastic tricoepithelioma [2,3,21] (Table 1). Biomedicines 2017, 5, 71 5 of 12

Biomedicines 2017, 5, 71 5 of 12

Merkel cell carcinoma is characterized by deeply basophilic tumoral cells with small-to-medium-sized cells, sparse cytoplasm, with dense round nuclei (Figure5). Tumor cells are arranged in a diffuse, trabecular, and/or nested pattern, usually involving the subcutis. Contrariwise, BCC shows a more asymmetrical architecture, palisading of peripheral tumor cells, and a typical cleft between tumorFigure cells 4. Basosquamous and stroma carcinoma (absent (hematoxylin in Merkel and cell eosin, carcinoma) 20×). [2,3] (Table1).

Figure 5. Typical small-to-medium-sized basophilic tumoral cells in a Merkel cell carcinoma. Figure 5. Typical small-to-medium-sized basophilic tumoral cells in a Merkel cell carcinoma. (hematoxilin and eosin, 30×). (hematoxilin and eosin, 30×). Clinical and histopathological differentiation between morpheaform BCC and trichoepithelioma Clinical(above and all histopathological desmoplastic tricoepithelioma) differentiation is usuall betweeny a frequent morpheaform problem (Figure BCC 6). BCC and is trichoepithelioma often an asymmetrical lesion, while trichoepithelioma is symmetrical, horn cysts are more frequent in (above alltrichoepithelioma, desmoplastic tricoepithelioma)the ulceration is frequent is in usually BCC and a rare frequent in trichoepithelioma problem (Figure[2,3]. Besides,6). BCCrims is often an asymmetricalof collagen lesion, bundles, while calcification, trichoepithelioma follicular/sebaceous/infundibular is symmetrical, differentiation, horn cysts and are cut more artefacts frequent in trichoepithelioma,are usually the present ulceration in trichoepithelioma, is frequent inwhile BCC they and are rare not present in trichoepithelioma in BCC. Morpheaform [2,3]. BCC Besides, rims of collagenexpress bundles, usually calcification, epithelial cell follicular/sebaceous/infundibular adhesion molecule (EPCAM) (BerEP4), cytokeratin differentiation, 6 (CK6, and that cutis artefacts negative in tricoepithelioma), Ki-67 (negative in trichoepithelioma), and androgen receptor (AR) [19– are usually21]. present Finally, in CD10+ trichoepithelioma, is positive in BCC while tumor they cells are and not positive present in the in BCC.stroma Morpheaformin tricoepithelioma. BCC express usually epithelialcytokeratin cell 20 (CK20) adhesion is present molecule in tricoepithelioma, (EPCAM) as (BerEP4), well as p75 cytokeratin and PHLDA1 6 are (CK6, positive that mainly is negative in tricoepithelioma),in desmoplastic Ki-67 tricoepithelioma (negative in trichoepithelioma),[2,3,21] (Table 1). and androgen receptor (AR) [19–21]. Finally, CD10+ is positive in BCC tumor cells and positive in the stroma in tricoepithelioma. cytokeratin 20 (CK20) is present in tricoepithelioma, as well as p75 and PHLDA1 are positive mainly in desmoplastic tricoepithelioma [2,3,21] (Table1). Microcystic adnexal carcinoma can be also confused with a BCC, positive pattern for the expression of carcinoembryonic antigen and epithelial membrane antigen, demonstrating follicular and eccrine differentiation and negative stain for Ber-Ep4 [2,3]. Histologically microcystic adnexal carcinoma is characterized by cords and nests of bland keratinocytes, keratin cysts, presence of a ductal differentiation, and a dense collagenous stroma [2,3] (Table1).

2.2. The Histologic Features of Actinic Keratosis, Bowen’s Disease, and Squamous Cell Carcinoma

2.2.1. General Considerations SCC accounts approximately for 20% of all cutaneous . In addition, SCC accounts for the most of NMSC-related metastatic disease and death [1]. Furthermore, SCC is reported to be within the top five most costly cancers in the United States [1]. In the future, SCC incidence will drastically increase, due to an increment in sun exposure, intensifying UV exposure, advancing age of the US population, enhanced public awareness of skin cancer, and more frequent skin examinations by physicians [1]. Cigarette smoking, therapeutic UV light exposure (including PUVA therapy and tanning Biomedicines 2017, 5, 71 6 of 12 bed use), HPV (types 16, 31 and 34), arsenic ingestion, polycyclic aromatic hydrocarbon exposure, immunosuppression, diets high in fat and meat, preexisting chronic dermatoses, and or sinus tract formation have been reported as risk factors [18–23]. Cutaneous conditions associated with the development of SCC include , erythematosus, , , epidermolysis bullosa, ab igne, chronic atrophicans, , dissecting of the , chromoblastomycosis, port wine stain, arteriovenous malformation, sebaceus, linear epidermal nevus, granuloma inguinale, fibroepithelial polyps, conglobata, , verruciform , and lymphogranuloma venereum [23–28]. Biomedicines 2017, 5, 71 6 of 12

Figure 6. Sometimes the differential diagnosis between a morpheaform BCC and a desmoplastic Figure 6. Sometimestricoepithelioma. the Rims differential of collagen diagnosisbundles surroundi betweenng basaloid a morpheaform cells without peripheral BCC palisading and a desmoplastic tricoepithelioma.and without Rims the of typical collagen cleft of bundles BCC, in a desmoplastic surrounding tricoepithe basaloidlioma cells (hematoxilin without and peripheral eosin, 10×). palisading and without the typical cleft of BCC, in a desmoplastic tricoepithelioma (hematoxilin and eosin, 10×). Microcystic adnexal carcinoma can be also confused with a BCC, positive pattern for the expression of carcinoembryonic antigen and epithelial membrane antigen, demonstrating follicular Deeperand skin eccrine invasion differentiation is associated and negative with stain increased for Ber-Ep4 metastatic [2,3]. Histologically disease microcystic and recurrence adnexal rates [21]. carcinoma is characterized by cords and nests of bland keratinocytes, keratin cysts, presence of a Lesions greaterductal than differentiation, 2 cm in and diameter a dense collagenous are twice stroma as [2,3] likely (Table to 1). recur and three times as likely to metastasize [19]. Tumor thickness may be measured in millimeters [21]. Peri-neural invasion may be 2.2. The Histologic Features of Actinic Keratosis, Bowen’s Disease, and Squamous Cell Carcinoma present, and it reduces the five-year survival rate [1,25]. In general terms, these tumors are larger at presentation2.2.1. (>2 General cm in diameter),Considerations less differentiated, and are associated with lymphadenopathy [17]. Generally, metastaticSCC accounts diseaseapproximately is uncommon for 20% of all cutaneous [1,21]. The malignancies. metastatic In addition, overall SCC rate accounts is about 2%, but it is betweenfor 10% the most and of 15% NMSC-related for lesions meta onstatic the disease lips and and death ears [1]. [ 1Furthermore,,19]. Eighty-five SCC is reported percent to be of metastases within the top five most costly cancers in the United States [1]. In the future, SCC incidence will affect only thedrastically lymph increase, nodes, due but to involvementan increment in sun of lungs,exposure, liver, intensifying bones, UV brain, exposure, and advancing mediastinum age may also occur [1,21]. of the US population, enhanced public awareness of skin cancer, and more frequent skin examinations by physicians [1]. Cigarette smoking, therapeutic UV light exposure (including PUVA 2.2.2. Actinictherapy Keratosis and tanning bed use), HPV infection (types 16, 31 and 34), arsenic ingestion, polycyclic aromatic hydrocarbon exposure, immunosuppression, diets high in fat and meat, preexisting chronic Amongdermatoses, non-melanoma and ulcer or skin sinus cancertract formation (NMSC), have been squamous reported as cellrisk factors carcinoma [18–23]. Cutaneous (SCC) accounts for the majorityconditions of NMSC-related associated with metastases the development and of death.SCC include A porokeratosis, wide diversity , of SCC subtypes exist, lichen planus, lichen sclerosus, epidermolysis bullosa, , acrodermatitis chronic several of whichatrophicans, are associatedichthyosis, dissecting with markedlycellulitis of th moree scalp, aggressive chromoblastomyco behaviorsis, port and wine other stain, with lower aggressive behaviorarteriovenous (Table malformation,2). Hoverer, nevus belowsebaceus, we linear report epidermal the main nevus, histologic granuloma variantinguinale, that usually we encounterfibroepithelial during the polyps, daily clinicalacne conglobata, practice: necrobiosis actinic keratosis, lipoidica, Bowen’sverruciform disease, xanthoma, invasive and SCC, and lymphogranuloma venereum [23–28]. keratoacanthomaDeeper (Table skin2). invasion is associated with increased metastatic disease and recurrence rates [21]. Lesions greater than 2 cm in diameter are twice as likely to recur and three times as likely to metastasize [19]. Tumor thickness may be measured in millimeters [21]. Peri-neural invasion may be present, and it reduces the five-year survival rate [1,25]. In general terms, these tumors are larger at presentation (>2 cm in diameter), less differentiated, and are associated with lymphadenopathy [17]. Generally, metastatic disease is uncommon [1,21]. The metastatic overall rate is about 2%, but it is between 10% and 15% for lesions on the lips and ears [1,19]. Eighty-five percent of metastases affect Biomedicines 2017, 5, 71 7 of 12

Table 2. Main histological features and differential diagnoses of non-melanoma skin cancers with squamous differentiation.

Differential Clinico-Pathological Features Diagnosis AK Atypical keratinocytes confined on basal layer. Bowen Atypical keratinocytes at every layer of epidermis. Symmetrical and circumscribed proliferation of keratinocytes, with central horn plug, with epidermis that extends over the tumor. Highly KA differentiated SCC. Invasive SCC Atypical and pleomorphic keratinocytes, involving the dermis and the sub-cutis with a potential metastatic spread. QE As Bowen, but in the mucosa. ACs Squamous differentiation, but does not show connection with the epidermis and highlights adnexal features. AD SCC Mixed glandular and squamous differentiation. VSCC * Exophytic squamous proliferation with marked papillomatosis and low atypia and the presence of -like changes SCC of the foot. Histologically is characterized hyperkeratosis, acanthosis with an undulating, densely keratinized, well differentiated EC squamous , deeply penetrating the soft tissues. IFK Sharply circumscribed endophytic verrucous proliferation with prominent squamous features. SK Acanthosis, absence of atypia, pseudo-horn cysts, in inflamed lesions, mitoses may be present. BP Atypical keratinocytes and mitoses. Histology similar to Bowen’s disease. Personal medical history of the patient, nodular proliferation without connection to epidermis, immunohistochemical evaluation. AK: actinic keratosis; KA: keratoacanthoma; SCC: squamous cell carcinoma; QE: Queyrat’s erytroplasia; AC: adnexal carcinomas; IFK: inverted follicular keratosis; SK: ; BP: ; AD SCC: adenosquamous carcinoma; VSCC: Verrucous squamous cell carcinoma. * When present in ano-genital region is also known with the term of Buschke–Löwenstein tumor; EC: epithelioma cuniculatum. Biomedicines 2017, 5, 71 7 of 12

only the lymph nodes, but involvement of lungs, liver, bones, brain, and mediastinum may also occur [1,21].

2.2.2. Actinic Keratosis Biomedicines 2017, 5, 71 8 of 12 Among non-melanoma skin cancer (NMSC), squamous cell carcinoma (SCC) accounts for the majority of NMSC-related metastases and death. A wide diversity of SCC subtypes exist, several of Actinicwhich keratosis are associated (AK) can with be markedly considered more as aggres an earlysive behavior SCC, rather and other than with a pre-malignant lower aggressive lesion [1,21]. behavior (Table 2). Hoverer, below we report the main histologic variant that usually we encounter AK couldduring potentially the daily evolve clinical into practice: invasive actinic tumor ke [ratosis,1,21,28 Bowen’s]. Pathologically, disease, invasive AK is characterizedSCC, and by a horizontalkeratoacanthoma alteration of parakeratotic (Table 2). and orthokeratotic hyperkeratosis, with an atrophic or acanthotic epidermis (FigureActinic7 )[keratosis21]. Neoplastic(AK) can be considered keratinocytes as an early in theSCC, basal rather layerthan a showpre-malignant increased lesion cellularity, nuclear pleomorphism,[1,21]. AK could andpotentially scattered evolve mitoses into invasive [21]. tumor A superficial [1,21,28]. Pathologically, lichenoid or AK perivascular is characterized lymphocytic by a horizontal alteration of parakeratotic and orthokeratotic hyperkeratosis, with an atrophic or infiltrate isacanthotic often associated epidermis with (Figure plasma 7) [21]. cells Neoplastic [21]. In keratinocytes the papillary in the dermis, basal elastosislayer show is increased often present [21]. The maincellularity, histologic nuclear variants pleomorphism, of AK and are scattered the acantholitic mitoses [21]. andA superficial the pigmented lichenoid or perivascular ones. Acantholitic AK is characterizedlymphocytic infiltrate by hyperkeratosis, is often associated acanthosis, with plasmaand cells [21]. suprabasal In the papillary dermis, elastosis characterized is by atypical basaloften keratinocytes.present [21]. The Acantholyticmain histologic variants AK can of be AK misdiagnosed are the acantholitic for and other the acantholyticpigmented ones. as Acantholitic AK is characterized by hyperkeratosis, acanthosis, and suprabasal acantholysis Grover disease,characterized Darier by disease, atypical wartybasal keratinocytes. dyskeratoma, Acantholytic as well asAK seborrheic can be misdiagnosed keratosis withfor other acantholysis. Pigmentedacantholytic AK is characterized diseases as Grover by disease, pigment Darier disease, in the warty keratinocytes dyskeratoma, and as melanophages; well as seborrheic specifically, the melaninkeratosis is focally with acantholysis. increased Pigmented in basal keratinocytes. AK is characterized However, by melanin a pigment slight in melanocytic the keratinocytes proliferation may be presentand melanophages; [2,3]. The specifically, main differential the melanin diagnosis is focally increased are Bowen’s in basal disease keratinocytes. (atypia However, of keratinocytes a slight melanocytic proliferation may be present [2,3]. The main differential diagnosis are Bowen’s at every layerdisease of (atypia the epidermis)of keratinocytes and at every flat seborrheiclayer of the epidermis) keratosis, and that flat seborrheic is characterized keratosis, that by is acanthosis and papillomatosischaracterized with by acanthosis sharp usually and papillomatosis flat base with and sharp distinct usually lateral flat base margins, and distinct intraepithelial lateral horn pseudo-cysts,margins, and intraepithelial monotonous horn basaloid pseudo-cysts, tumor and cellsmono withouttonous basaloid atypia tumor [21 ].cells without atypia [21].

Figure 7.FigureActinic 7. Actinic keratosis keratosis with with Bowenoid Bowenoid fe featuresatures (hematoxylin (hematoxylin and eosin, and 40×). eosin, 40×).

2.2.3. Main Histologic Variant and Differential Diagnoses of NMSC with Squamous Differentiation Bowen’s disease can be defined as an intra-epidermal carcinoma with atypia of keratinocytes at every layer of the epidermis (squamous cell ) [21]. Pathologically, it is characterized by and by bulbous rete ridges in the epidermis [21]. In addition, the entire epidermis shows disordered maturation with atypical keratinocytes, individual cell keratinization, pleomorphic nuclei, atypical mitoses, multi-nucleated tumor-cells, and an intact basal layer [21]. A lymphocytic perivascular or lichenoid infiltrate is present, often with mixed plasma cells [21]. The main histological differential diagnoses are erytroplasia of Queyrat (identical to Bowen’s disease but on mucosa), Paget’s disease (pagetoid distribution of clear tumor cells in epidermis, with CAM5.2+ and CK7+ cells), Bowenoid papulosis (genital HPV infection with atypical keratinocytes and mitoses identical microscopically to Bowen’s disease), superficial spreading melanoma (pagetoid intraepidermal spread of atypical in solitary elements or in nests, positive to S100, HMB45), and irritated seborrheic keratosis (absence of keratinocyte atypia, intense inflammation, and presence of mitoses due to the marked inflammation) [21] (Table2). In the current review, we also included keratoacanthoma as well since it is an epithelial with a squamous differentiation. Keratoacanthoma is mainly divided into keratoacanthoma Biomedicines 2017, 5, 71 9 of 12 centrifugum marginatum (giant keratoacanthoma with striking centrifugal spread) and multiple [2,3]. Histopathologically, it is characterized by a symmetrical and circumscribed proliferation of keratinocytes, with a central horn plug, epidermis that extends over the tumor, and pale ground-glass-like keratinocytes with uniform differentiation [2,3]. The main histologic differentiation is the invasive SCC (Table2). Keratoacanthoma can be considered a highly differentiated SCC, which only rarely escapes intrinsic tumor surveillance [2,3]. SCC is mainly divided in conventional, spindle-cells, verrucous, acantholytic, and lympho-epithelioma like SCC. The Broders classification is based on the degree of tumor differentiation [21]. More specifically, grade I includes tumors composed of <25% undifferentiated cells, grade II lesions with <50% undifferentiated, grade III lesions with <75% undifferentiated cells, and grade IV lesions with >75% undifferentiated cells [21]. Conventional SCC is characterized by atypical cells in the dermis, showing enlarged and pleomorphic nuclei with atypical mitotic activity [21] (Table2). Inflammation is usually present in ulcerated lesions and typically consists in lymphocytes, plasma cells, and neutrophils [1]. Keratinous pearls are typically present on the dermis, surrounded by nests of atypical cells and reduced stroma with lymphocytes [1]. The conventional SCC may also show desmoplastic features [21]. Desmoplastic SCC is characterized by aggregates of moderate or poorly differentiated keratinocytes surrounded by desmoplastic stroma, which by definition includes at least 30% of the tumor volume (Figure8)[ 21]. This variantBiomedicines is associated 2017, 5, x FOR with PEER peri-neural REVIEW invasion and higher metastatic spread [21].10 of 12

Figure 8.FigureDesmoplastic 8. Desmoplastic squamous squamous cell cell carcinomacarcinoma (hematoxylin (hematoxylin and eosin, and 10×). eosin, 10×).

Adenosquamous carcinoma is an uncommon variant, characterized by the simultaneous Spindlepresence cell SCCof mixed is associated glandular and with squamous previous differentiation, trauma or with radiotherapy an aggressive and clinical it is behavior. the main variant associatedHistologically, with the Marjolin it is characterized ulcer (post-traumatic by the presence ulcerated of features SCC)of SCC [ 21with].It intercellular is characterized bridging, by spindle keratin pearl formation, parakeratotic differentiation, and an adenomatous component characterized cells that involve the dermis and intermingle with strands of collagen in a whorled fashion [29]. by the presence of intracytoplasmatic mucin, although in the presence of true glandular formation, The surroundingthe presence stroma of intra-cytoplasmatic may be myxoid mucin is and not required pleomorphic to reach gianta diagnosis cells [34] may (Table be 2). appreciated, but desmoplasia shouldFinally, verrucous not represent carcinoma more of the than skin and 30% mu ofcosa tumor is a well-differentiated volume [21]. SCC, Mitotic which activitywhen is also present withpresent atypical in the genito-anal mitoses [ 26region]. A is possible also known subtype as the Buschke-Löwenstein of SCC is pigmented tumor [35]. SCC Histologically, [21]. This subtype is it is characterized by exophytic squamous proliferation with marked papillomatosis and low atypia rare and preferentiallyand the presence affects of koilocyte-like the oral changes, and conjunctival central collection mucosa of neutrophils in dark-skinned may be also patientspresent [35]. [19 ]. AcantholiticThere is a SCC lot of (ASCC) confusion is regarding also known the terminology as of this rare tumor; [1,29 it– 32was]. variously ASCC is described usually found on the head andas giant neck condyloma in elderly acuminate, people, squamous above all papillomatosis, in post-radiotherapy condyloma sitesacuminata [21]. with ASCC malignant is characterized by an adenoidtransformation, pattern, and with well-differentiated a lot of dyskeratotic squamous cells cell [ 1carcinoma.–3,33]. ASCC Howeve isr, similar verrucous to eccrinecarcinoma neoplasms, presents as a distinct entity with exo-endophytic growth pattern (in contrast to condyloma but it is PAS,accuminata) negative offor squamous carcinoembryonic cells, showing antigen mild atypia and with positive pushing for margins epithelial (in membranecontrast to the antigen [19]. A variant ofinvasive ASCC character is the pseudo-vascular of well-differentiated adenoid squamous SCC, carcinoma) which [30]. shows Finally, less epithelioma acantholysis cuniculatum and a preminent glandularis appearance a rare variant [of1 ].SCC This of the tumor foot. Histologically, is characterized it is characterized by pseudovascular hyperkeratosis; lumen-like acanthosis with features with connectionan toundulating, the overlying densely keratinized, epidermis well and differentiated tumor cells squamous that dissectepithelium, through deeply penetrating dermal collagen the and soft tissues; frequently separated by sinuses filled with inflammatory cells. Its is around adnexalunknown, structures although it [1 may]. The be preceded pseudo-vascular by a plantar adenoidwart [36]. SCC cells show also hobnail features [1]. Generally, the main histological differential diagnoses of SCC are metastasis from internal squamous cell carcinoma (importance of the personal medical history of the patient, nodular proliferation without connection to epidermis, immunohistochemical evaluation), adnexal carcinoma (adnexal carcinoma may have squamous differentiation, but does not show connection with the epidermis and highlight adnexal features), keratoacanthoma (a highly differentiated SCC), and inverted follicular keratosis (sharply circumscribed endophytic verrucous proliferation with prominent squamous features) [2,3] (Table 2).

Acknowledgments: We thank our patients for their cooperation. The authors would also like to acknowledge colleagues who have contributed in different aspects of this study such as Giulia Macrì, Sonia Tofani, and Carlo Drago for their contribution.

Conflicts of Interest: The authors declare that there is no conflict of interest regarding the publication of this article. Biomedicines 2017, 5, 71 10 of 12

Finally, papillary SCC is more frequent in elderly women and in immunosuppressed patients; it is characterized by red to tan features [33]. Adenosquamous carcinoma is an uncommon variant, characterized by the simultaneous presence of mixed glandular and squamous differentiation, with an aggressive clinical behavior. Histologically, it is characterized by the presence of features of SCC with intercellular bridging, keratin pearl formation, parakeratotic differentiation, and an adenomatous component characterized by the presence of intracytoplasmatic mucin, although in the presence of true glandular formation, the presence of intra-cytoplasmatic mucin is not required to reach a diagnosis [34] (Table2). Finally, of the skin and mucosa is a well-differentiated SCC, which when present in the genito-anal region is also known as the Buschke-Löwenstein tumor [35]. Histologically, it is characterized by exophytic squamous proliferation with marked papillomatosis and low atypia and the presence of koilocyte-like changes, central collection of neutrophils may be also present [35]. There is a lot of confusion regarding the terminology of this rare tumor; it was variously described as giant condyloma acuminate, squamous papillomatosis, condyloma acuminata with malignant transformation, and well-differentiated squamous cell carcinoma. However, verrucous carcinoma presents as a distinct entity with exo-endophytic growth pattern (in contrast to condyloma accuminata) of squamous cells, showing mild atypia with pushing margins (in contrast to the invasive character of well-differentiated squamous carcinoma) [30]. Finally, epithelioma cuniculatum is a rare variant of SCC of the foot. Histologically, it is characterized hyperkeratosis; acanthosis with an undulating, densely keratinized, well differentiated squamous epithelium, deeply penetrating the soft tissues; frequently separated by sinuses filled with inflammatory cells. Its pathogenesis is unknown, although it may be preceded by a plantar [36]. Generally, the main histological differential diagnoses of SCC are metastasis from internal squamous cell carcinoma (importance of the personal medical history of the patient, nodular proliferation without connection to epidermis, immunohistochemical evaluation), adnexal carcinoma (adnexal carcinoma may have squamous differentiation, but does not show connection with the epidermis and highlight adnexal features), keratoacanthoma (a highly differentiated SCC), and inverted follicular keratosis (sharply circumscribed endophytic verrucous proliferation with prominent squamous features) [2,3] (Table2).

Acknowledgments: We thank our patients for their cooperation. The authors would also like to acknowledge colleagues who have contributed in different aspects of this study such as Giulia Macrì, Sonia Tofani, and Carlo Drago for their contribution. Conflicts of Interest: The authors declare that there is no conflict of interest regarding the publication of this article.

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