While numerous subtypes of BCC have been described according to their microscopic appearance, most of these variants will demonstrate little significant difference in biological behaviors, with minimal competence for distant spread. In contrast, there exists a wide histopathologic diversity of SCCs, many of which are associated with markedly different clinical behaviors. These can range from indolent tumors with low metastatic potential, to remarkably aggressive tumors with high invasive potential [6–9]. The ability to distinguish between these variants microscopically is thus critically important in the clinical diagnosis and treatment of SCC, with early treatment of high-risk tumors resulting in better patient outcomes with a lower risk of tumor metastasis and recurrence [4, 10].

The purpose of this review is thus two fold. First, we aim to provide a clear and comprehensive means of discriminating between various SCC lesions on the basis of intrinsic differences in their histopathology. This will include a detailed description of the unique histopathological features pertaining to each SCC subtype, highlighting the often subtle differences which can be used in their distinction. More specifically, our descriptive analysis can be loosely organized into three separate categories and will encompass: actinic or solar keratoses (AKs) and SCC in situ (Bowen's disease), common precursors to SCC formation seen as a direct result of excess sun exposure; invasive SCC (SCCI), clear-cell SCC, spindle cell (sarcomatoid) SCC, and SCC with single cell infiltrates, tumor subtypes which emerge from the invasive progression of the aforementioned lesions; de novo SCC, lymphoepithelioma-like carcinoma of the skin (LELCS), and verrucous carcinoma (VC), highly uncommon SCC variants with no direct correlation to sun exposure or actinic precursors. While there are undoubtedly several other rare subtypes of SCC and squamoid neoplasms which we will not be addressing directly, the vast majority of commonly seen SCCs can be incorporated into one of the categories mentioned above.

Clinically, AKs will manifest as ill-marginated, erythematous, scaling, and rough papules or patches. These will typically be found in areas displaying other signs of solar damage, such as atrophy, uneven pigmentation, and telangiectasias [14]. Lesions are further associated with three possible clinical outcomes, which include spontaneous regression, persistence as a benign AK, or evolution into an invasive SCC (SCCI) [15]. Although the overwhelming majority of SCCs are found to be associated with AKs [16], these lesions are generally considered to have a low malignant potential, with only 5%–10% progressing to SCCI over the next several years [17].

Histopathologically, several different variants of AKs have been identified, including hypertrophic, atrophic, acantholytic, pigmented, proliferative, and Bowenoid subtypes. In general, the hypertrophic and proliferative variants are associated with more aggressive biologic behaviors and have a higher malignant potential [7, 9, 18, 19].

By definition, AKs are confined to foci within the epidermis. They are associated with aggregates of atypical, pleomorphic keratinocytes which show nuclear atypia, dyskeratosis, and loss of polarity. Hyperkeratosis and parakeratosis are often seen, the latter overlying the abnormal cells in the epidermis. Due to the sparing of segments of the epithelium overlying adnexal structures, a characteristic pattern of alternating orthokeratosis and parakeratosis, referred to as the “flag-sign,” can often be seen (Figure 1(a)) [20]. Atypical keratinocytes from the basal cell layer will frequently extend into the granular and cornified layers, however, they will not span the full thickness of the epidermis (Figure 1(b)) [20, 21]. The exception to this criterion is the Bowenoid variant of AK, which resembles Bowen's disease but is less disordered with less nuclear atypia and crowding [9]. The basal layer in AKs will often appear to be more basophilic than normal, which is generally thought to be a consequence of the close crowding of atypical keratinocytes (Figure 1(b)). Some cases will also show basal layer degeneration and the formation of Civatte bodies, the result of a lichenoid infiltrate with irregular acanthosis. This can be distinguished from lichenoid dermatitis by the presence of keratinocyte atypia [22].

Bowen's disease may arise on the skin of any body site: however, the vast majority of cases (approximately 72%) are found on sun-exposed surfaces such as the head, neck, and hands [25]. Mucosal surfaces and the nail bed are also commonly involved. Only in rare circumstances will SCCS be found on the palms of the hand or the soles of the feet [26]. Bowen's disease often presents as an asymptomatic, erythematous, well-demarcated, scaly patch or plaque. It tends to be slowly enlarging, and usually has a fairly irregular border. Lesions may become hyperkeratotic, crusted, fissured or ulcerated, and can occasionally be pigmented, especially when found in the genital region and the nails [26].

Bowen's disease can be considered a low-grade form of SCC, with the majority of studies reporting the risk of progression to SCCI at 3%–5%. The risk of invasive development is estimated to be slightly higher (approximately 10%) for genital Bowen's disease, also known as erythroplasia of Queyrat [27]. Despite the low incidence of malignant progression, Bowen's disease is of significant consequence since approximately 20% of the tumors that do develop into SCCI will eventually become metastatic [28]. The association of Bowen's disease with other forms of malignancy such as internal cancers is a highly controversial subject and continues to be an area of active research [29].

Histopathologically, the epidermis in Bowen's disease will show hyperkeratosis and parakeratosis. There will also be marked acanthosis with elongation and thickening of the rete ridges. These changes will overly keratinocytic cells which are often highly atypical and may in fact have a more unusual appearance than SCCI (Figure 2(a)). The atypia spans the full thickness of the epidermis, with the keratinocytes demonstrating intense mitotic activity, pleomorphism, and greatly enlarged nuclei. They will also show a loss of maturity and polarity, giving the epidermis a disordered or “windblown” appearance. Two types of multinucleated cells may be seen: the first will present as a multinucleated giant cell, and the second will appear as a dyskeratotic cell engulfed in the cytoplasm of a keratinocyte [25]. Occasionally, cells of the upper epidermis will undergo vacuolization, demonstrating an abundant and strongly eosinophilic cytoplasm.

Histopathologically, SCCIs will frequently bear close resemblance to their precursor AK lesions, but can be distinguished from the latter via the presence of infiltrative cells passing through the basement membrane into the dermis (Figure 3) [6, 14]. This infiltrate can be somewhat difficult to detect in the early stages of invasion: however, additional indicators such as full thickness epidermal atypia and the involvement of hair follicles can be used to facilitate the diagnosis [31]. Later stages of invasion are characterized by the formation of nests of atypical tumor cells in the dermis (Figure 4), often with a corresponding inflammatory infiltrate.

Histopathologically, Kuo [34] subdivided clear-cell SCC into 3 different categories: keratinizing (Type I), nonkeratinizing (Type II), and pleomorphic (Type III). Type I lesions are characterized as sheets or islands of tumor cells with clear, empty-appearing cytoplasm (Figure 5(a)). Cytoplasmic growth will displace cell nuclei to the periphery, causing these cells to be indistinguishable from regular mature adipocytes. Some cells will have a “bubbled” cytoplasmic appearance, leading them to resemble sebaceous neoplasms. The former can be distinguished from the latter through the additional presence of foci of keratinization and keratin pearls (Figure 5(b)). The surrounding stroma will be fibrotic, with a sparse inflammatory infiltrate. Type II lesions are predominantly dermal in origin, with no clear connection to the overlying epidermis. They are characterized by parallel or anastomosing cords of tumor cells separated by a compressed, fibrotic stroma with a dense inflammatory infiltrate. Tumor cells will have central nuclei with a finely reticulated clear cytoplasm, and evidence of central necrosis within tumor cords may be present. Ductal or glandular differentiation does not occur, and unlike Type I there is no keratinization. Type III lesions arise from the epidermis and show extensive ulceration. Atypical clear-cells display marked nuclear pleomorphism, with foci of squamous differentiation, areas of acantholysis, and the presence of dyskeratotic cells within pseudoglandular spaces. Perineural and vascular space will show considerable invasion. Of importance to note is that in all three types described above, there is no evidence of either glycogen or mucin found within the tumor cells, and only trace amounts of lipid. This is consistent with the hypothesis that clear-cell changes are degenerative [34].

Histopathologically, spindle cell SCC may be almost entirely composed of atypical spindle cells arranged in a whorled pattern (Figure 6(a)), or may have a combination of spindle cells and more conventional SCC cells, often with an associated AK [37]. Unlike conventional SCC, however, tumor cells will singly infiltrate the dermis without the formation of nests or cords. Connection to the overlying epidermis can vary. Bizarre pleomorphic giant cells, as well as heterologous elements with numerous mitotic figures can be identified, often with deep infiltration into the dermis, subcutis, fascia, muscle, and even occasionally bone. The stroma, however, should not be significantly desmoplastic.

Unlike conventional SCCs, which tend to occur on the face, neck, hands, and other sun-damage prone areas, de novo SCCs are most commonly found on the lower extremities, where burn scars are more prevalent. Although females on average sustain a greater number of burn injuries than males, there is a predilection for malignant scar development in men, and de novo SCCs are significantly more likely to be found in males than in females [46]. They usually emerge 20 to 40 years after the original trauma occurred, and will present clinically as nonhealing, exophytic growths or indurated ulcers which have recently became painful and may have a foul smelling discharge [46].

Oroaerodigestive VC is thought to be the most common form of VC and accounts for anywhere between 2%–12% of all oral carcinomas [50]. It is generally seen in elderly Caucasian males on the gingival and buccal mucosa, but has been known to involve the larynx as well. Early lesions will appear as white, translucent, keratotic patches found on an erythematous base, and will subsequently develop into soft, rubbery papillary growths with pebbly surfaces. Ulceration and fistulation are also commonly seen, with occasional local invasion into soft tissue and bone. Oroaerodigestive VC has strong positive associations with both low- and high-risk HPV, with high-risk HPV presenting in up to 45% of laryngeal VC cases [51]. It has also been linked to chemical carcinogens such as chewing tobacco and snuff [52], as well as other conditions affecting the oral mucosa such as lichen planus, chronic candidiasis, leukoplakia, and chronic lupus erythematosus [53].

Anourogenital VC is thought to account for approximately 5%–24% of all penile carcinomas [54], and typically presents on the glans penis and prepuce of middle-aged uncircumcised males. It is only rarely found in the female genital tract. Lesions will appear large, exophytic, and cauliflower-like, with verrucous or ulcerated surfaces. This variant of VC is most commonly associated with low-risk HPV, with a positive correlation found in up to 50% of cases, but it can be seen in association with high-risk types as well [54].

Palmoplantar VC is generally found on the soles of elderly Caucasian males, but may also be seen on the toes, the heel, or the dorsum of the foot [55]. Lesions are often initially mistaken for plantar warts, however, they tend to slowly evolve into bulky exophytic masses with ulceration and foul-smelling discharge. It is thought to be associated loosely with the low-risk HPV types, but is also related to trauma, chronic irritation, and other forms of HPV infection [49].

VC lesions found on other cutaneous sites are rare occurrences, and tend to appear as slow-growing warty or cauliflower-like lesions with only weak associations to HPV infections [49].

Despite these separate subclassifications, all four forms of VC share a similar histopathological description. The epithelium will generally display a characteristic endoexophytic growth pattern, with a prominent granular layer showing marked hyperkeratosis and parakeratosis, as well as acanthosis and papillomatosis (Figure 8(a)). Keratinocytes are often enlarged with prominent nuclei, but atypia is otherwise minimal. The epidermis remains well differentiated with obvious stratification, and will demonstrate broad, bulbous, rete-like projections which will descend deep into the underlying dermis (Figure 8(b)). These will generally be surrounded by a dense inflammatory infiltrate, and can often result in the formation of sinuses and keratin-filled cysts [47]. The projections have a remarkably indolent appearance, and lack all the classical signs of invasion such as cellular atypia and infiltration. Malignant changes are therefore the result of compressive destruction rather than invasion, and the margins of VC lesions will often show aggressive borders which may extend into the adjacent structures causing destruction of the local connective tissue, muscle, cartilage, or bone [49].

Histopathologically, islands or syncytial sheets of tumor will be seen in the mid-to-deep dermis. These are composed of large, pale, eosinophilic polyhedral cells, with vesicular nuclei and prominent nucleoli (Figure 9(a)). The cells tend to be cohesive and atypical, exhibiting numerous mitoses and lacking any distinct cellular borders. Additionally, the presence of an immune infiltrate will be visible surrounding the atypical aggregates, although the degree to which infiltration occurs has been known to show marked variation. For instance, while the immune response can be sparse and peripherally located, it can also be extremely dense, obscuring the nearby tumor cells. This infiltrate is composed largely of plasma cells and small lymphocytes, however the occasional neutrophil and eosinophil can also be found [59].

Size and depth of invasion are perhaps the most important determinants of the likelihood of tumor recurrence and metastasis [33]. As a broad rule, tumors less than 2cm in size will only rarely metastasize and are unlikely to recur whereas those greater than 2cm in size pose a significant threat of metastasis and recurrence [63]. Similarly, tumors that exceed 4mm in depth or Clark's level III, showing involvement of both the deeper levels of the dermis as well as the subcutaneous tissue, have a much more aggressive course of action and over a seven-fold increase in the probability of metastasis [64, 65]. Skin lesions greater than 8 mm in depth or Clark's level V pose such a significant threat of metastasis that nodal involvement and prophylactic node dissection should be seriously considered [65]. As expected, SCCI, which is derived largely from a superficial precursor lesion, is considered to be an indolent tumor. In contrast, SCC with single cell infiltrates, which is defined predominantly by dermal activity, is considered to be a more aggressive tumor type.

The degree of histologic differentiation, as well as the anatomic site of the lesion, will also play a role in SCC evaluation and prognosis. Poorly differentiated tumors, particularly from the ear or the lip, will be three times more likely to metastasize, and twice as likely to recur when compared to tumors that are well differentiated [33]. Despite this, the majority of metastatic tumors will be moderately to well differentiated, highlighting the importance of assessing all the prognostic factors in the evaluation of metastatic potential [33].

Another essential component in assessing the malignant potential of a tumor is the presence of perineural and perivascular spread. Perineural involvement (PNI) is thought to occur in approximately 14% of all SCC tumors arising on the head or neck [66], and is indicative of the inherently aggressive nature of the tumor. Accordingly, tumors with PNI will show a much greater likelihood of local recurrence (23%) relative to those without (9%) [67]. They will also be associated with a worse overall outcome, and a significant increase in the disease-specific mortality rate. Clinically, perineural involvement can present as conventional SCC with an associated numbness, facial muscle weakness, twitching, or visual change. However, there are often no clinical symptoms of nerve involvement, and PNI is most frequently diagnosed microscopically [66, 67].

Similarly, invasion of capillary lymphatics signifies a more aggressive tumor nature and is correlated with an increased incidence of metastases, local recurrence, and disease-specific death [67]. Additionally, SCC metastasis occurs predominantly via local lymphatics and often deposits in the lymph nodes of the neck [68]. Although invasion tends to remain localized to regional nodes, prognosis remains extremely poor with only a 34.4% cure rate [33].

Histopathologically, PNI and perivascular invasion will appear as an overlying SCC with atypical tumor cells which have penetrated the nerve or vascular tissue (Figure 10). This can present in a variety of different invasion patterns, most frequently involving a complete encircling of the nerve or vessel by tumor cells. An incomplete, crescent-like pattern of atypical cells is also commonly seen. Occasionally, tangential contact, permeation, and lamination can be observed. Invasion almost always occurs contiguous to the main body of the tumor; however, it has been known on occasion to affect more distant nerve and vascular sites. Usually, tumor cells arranged in solid or sheet-like patterns are less invasive, and will pass around the nerve or vessel. In contrast, individual tumor cells will generally penetrate and track along associated structures [66, 67].

Finally, host immunosuppression can greatly increase the likelihood of SCC development, recurrence, and malignant spread [69–72]. Suppression may be due to an underlying malignancy, the active use of immunosuppresive agents during transplant therapy, or infection with HIV. In fact, NMSC is considered to be one of the most common side effects of long-term immunosuppressant use seen in transplant recipients. Unlike the general population, these patients are more likely to present with SCCs than BCCs [73, 74]. These lesions will typically emerge on the sun-exposed surfaces of the body, and will be found more frequently in patients with histories of sun exposure [75] and HPV infection [76]. Immunosuppressed patients will usually present with multiple SCC lesions, and while the risk of each individual lesion metastasizing is not markedly elevated, the presence of so many will have the effect of increasing the overall risk of metastasis [77, 78].