Histology for Pathologists

Editors: Mills, Stacey E.

Title: Histology for Pathologists, 3rd Edition

Copyright 2007 Lippincott Williams & Wilkins

> Table of Contents > VII - Alimentary Tract > 27 - Anal Canal

27

Anal Canal

Claus Fenger

Introduction

The anal canal has a complex anatomy and histology, and new information still turns up with regard to its embryology, structure, function, and pathology. Unfortunately, much confusion continues about definitions and nomenclature. This chapter therefore includes a historical review and a discussion of the terms used for the different structures (1).

Historical Review

Anal diseases and their treatment are mentioned as far back as the Egyptian papyri (2). Nevertheless, there are few early descriptions of anal anatomy. Of note is Galenos (130 200 AD), who compared the anus to a laced-up purse (3). The first observations on the anal canal mucosa were published by Glisson (1597 1677) (4), who noted the anal valves. In 1717, Morgagni (5) mentioned the anal columns and included the now famous drawing in the Adversaria (Figure 27.1); in addition, it shows pronounced papillae. In 1727, Heister (6) described the smooth zone between the anal valves and the perianal skin; and, in 1732, Winsl w (7) described the semilunar lacunae between the valves and the bases of the anal columns.

The first exact microscopic description of the different epithelial zones in the anal canal was given by Robin and Cadiat in 1874 (8). The perianal apocrine glands were found by Gay in 1871 (9), and in 1878 Chiari introduced the theory of the anal sinus infection in the pathogenesis of anal fistulas (10). Hermann and Desfosses' (11) microscopic description of anal glands followed shortly afterward.

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In 1877 Hilton (12) introduced the term white line for the junction between the skin and the mucous membrane and corresponding to the linear interval between the internal and external sphincter muscle. In 1896, Stroud (13) introduced the term pecten for the smooth area between the anal valves and Hilton's white line. However, later investigators recommended that use of the term Hilton's white line should be discontinued because no anatomic feature identified it (14,15). Detailed macroscopic and stereomicroscopic investigations of surgical specimens of the anal canal have not shown any such structure (16,17).

The term anal canal was proposed by Symington in 1888 (18) for what had earlier been described as the third or perineal part of the rectum, that is, the part extending from the level of the pelvic floor backward and downward to the anal opening. This definition corresponds to the surgical anal canal, whereas the term anatomic anal canal has been used for the area between the line of anal valves and sinuses [dentate line (DL)] and the anal verge alone (Figure 27.2). The anal verge can be defined as the point (line) where the walls of the anal canal come in contact in their normal resting state (19).

As to embryology, Tourneux in 1888 (20) and Retterer in 1890 (21) wrote that the cloacal membrane was divided into anal and urogenital membranes by a descending septum, thus giving basis for the often quoted but never illustrated delusion that the DL is the site of a former anal membrane.

Embryology

In the early embryo (Carnegie stage 13, crown-rump length 5 mm), the primitive urogenital sinus and anorectal canal are separated by the urorectal septum and open into a common cloaca, which is closed at its ventral surface by the cloacal plate. As the embryo grows and its caudal curvature decreases, the distance between the urorectal septum and the cloacal plate decreases, but these two structures never fuse.

Due to apoptosis, the dorsal part of the cloacal plate gradually becomes more like a membrane, which ruptures about 49 days postfertilization (Carnegie stage 19, crown-rump length 16 18 mm). Soon after, a secondary occlusion of the anorectal canal occurs as a result of cell adhesion and formation of an epithelial plug at the level of the anal orifice. Recanalization takes place at 30 to 35 mm crown-rump length, again due to apoptosis. The cloaca itself does not contribute to the formation of the anus or rectum (22,23). The characteristic epithelium of the anal transitional zone is present already in the thirteenth week (Figure 27.3).

The prevalence of anal anomalies in Europe is 4 per 10,000 births, and two-thirds occur together with other anomalies. Most of the isolated anomalies are atresias with or without fistula, and 90% of these are located below the levator ani. The etiology to anal atresia is still under discussion (24).

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Nomenclature of the Anal Canal

It would seem natural to start with a definition of the anal canal; but, because there are several definitions and new terms are still introduced (25,26), a description of the anatomical landmarks and epithelial zones may be the best introduction to this never-ending discussion.

Official Terms

Official terms (27) for the structures seen on the anal canal surface include columnae anales, sinus anales, valvulae anales, linea pectinata, pecten analis, and linea anocutanea. However, the latter three terms are not generally accepted, and many synonyms have been used. Among the epithelial zones, only the anal transition(al) zone has got its own name.

When discussing anal nomenclature, one must always bear in mind that the terms anal columns, valves, and sinuses, and the line composed by the latter two structures are macroscopic landmarks. However, although they can be visualized by using macroscopic staining (28), the different zones in the anal canal are microscopic structures, and they do not correspond exactly to the macroscopic landmarks (16).

The most important macroscopic landmark is the line composed of the anal valves and sinuses and the bases of the anal columns (Figures 27.4,27.5). A list of names introduced for this line is given in Table 27.1 (1). Among these, the term dentate line is chosen by the International Union Against Cancer (UICC) and the World Health Organization (WHO) and is also widely used in textbooks (25,29,30,31,32). Anal valves and papillae are not present as often as reported in anatomy textbooks, and crypts and sinuses may be found over a larger area than compatible

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Epithelial Zones

Whatever definition of the anal canal is used, the sequence of epithelial zones in this area (i.e., rectum to perianal skin) follows (Figure 27.6):

• the zone covered with uninterrupted mucosa of colorectal type

• the zone with epithelial variants [anal transitional zone (ATZ)]

• the zone covered with uninterrupted squamous epithelium

• the perianal skin with keratinized squamous epithelium and skin appendages

These zones have been given many different names over the past century (Table 27.2) based on macroscopic, histologic, and embryologic considerations. Among the terms applied to the zone with epithelial variants, situated between the colorectal-type mucosa above and the squamous epithelium below, the most widely accepted term is now the anal transition(al) zone (28). The term zona columnaris refers to a macroscopic observation; the names membranous, cloacogenic, and junctional zones all indicate the site of the cloacal membrane in early fetal life and the meeting point of endoderm and ectoderm, a theory that was rejected decades ago. The terms intermediate or middle zone could be used, but neither gives any information about the character of the epithelial lining. Hemorrhoidal zone is misleading because hemorrhoids are not confined to this area.

The definition of the anal transitional zone is as follows. The ATZ is the zone interposed between uninterrupted colorectal mucosa above and uninterrupted squamous epithelium below, irrespective of the type of epithelium present in the zone itself (28).

From this it follows that names related to the histologic appearance also would be appropriate for the other zones. One can therefore use the term colorectal zone for the mucosa above the ATZ and squamous zone for the area below, which gradually merges into the perianal skin (Figure 27.6).

These zones are not always clearly visible to the clinician, and biopsy specimens should therefore be forwarded to the pathologist with an explanation of their location in relation to the DL. Biopsy samples from the lower part of the anal canal can be guided using a colposcope (Figure 27.7) (33,34).

Extent of the Zones

The colorectal zone, ATZ, and the area covered by squamous epithelium can easily be distinguished on longitudinal histologic sections (Figure 27.8). However, this method gives only limited information regarding the extent of the zones because the outlines are highly irregular. A better

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By using this method on a consecutive series of 113 anal canals, four main variants could be visualized. In most cases (88%), the ATZ started at the DL and extended on average 9 mm cranially (range: 3 20 mm) (Figure 27.9). In a few cases (7%), the ATZ started below the DL (Figure 27.10) or above the DL (4%) (Figure 27.11). In one case, the ATZ was totally absent (Figure 27.12). In addition, the method clearly demonstrated the highly irregular outlines of the ATZ, especially at the upper border, as visualized by stereomicroscopy (Figure 27.13) (17). Others, using slightly different techniques on 28 anal canals, have found the median length of the ATZ to be about 5 mm (35).

The squamous zone normally extends downward from the DL, but squamous epithelium is often found covering parts of the anal columns (17). The lower border of the squamous zone is difficult to determine because the squamous zone gradually merges into the perianal skin. If the transition is defined by the occurrence of skin appendages, the lower border of the squamous zone is located outside the anal canal. This is particularly the case when hemorrhoids or prolapse is present. From this it follows that the histologic transition to perianal skin does not necessarily correspond to the definitions of the lower border of the anal canal.

The perianal region is not well defined; but, for the purpose of tumor classification, a boundary located 5 to 6 cm from the transition to squamous mucosa is recommended by the American Joint Committee on Cancer (AJCC) (36). This might be inappropriate as the region thus includes the most posterior parts of the vulva or scrotum.

Definitions of the Anal Canal

Various definitions of the anal canal have been suggested through the ages. Some are based on macroscopic landmarks (Figure 27.2), others on the extent of the epithelial zones (Figures 27.2, 27.6). Of the two based on macroscopy, the anatomic anal canal, which extends from the DL down

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The histologic anal canal is defined by the extent of the special mucosa in this area (i.e., the ATZ and the squamous epithelium down to the perianal skin). Using this definition, microscopic identification of the canal is reasonably easy, and all the special tumors in this area have their origin in the canal. The drawback is that the extent of the anal canal cannot be estimated by the clinicians and that colorectal neoplasias can have their origin inside the histologic anal canal because it also harbors colorectal-type epithelium scattered in the ATZ.

The surgical anal canal can be identified easily by the clinician and described in the surgical report, thus making communication between surgeon and pathologist easier. The surgical anal canal is also regarded as a functional unit. However, with regard to the mucosal lining, the surgical anal canal most often includes a ring of more or less normal appearing colorectal mucosa at its upper border.

The lack of agreement between macroscopic and microscopic definitions is well known from other areas (i.e., the cervical transformation zone and the esophagogastric junction) and must be accepted. The essential point is that when the clinician describes the location of a process or a biopsy, the position in relation to the DL is the important message. The pathologist will then report what epithelial types are present in the material and, considering the above-mentioned variations, decide whether it is normal.

As the anal canal is described using the definition of the surgical anal canal by the WHO (31), as well as in the tumor-node-metastasis (TNM) system (32), this definition will also be used in the following text.

Anatomy

Mucosal Surface

Following the definition of the surgical anal canal (18), the structure extends from the level of the pelvic floor (pelvic visceral aperture, anorectal ring) to the anal opening (anal verge) (Figure 27.2) and in a living person has an average length of 4.2 cm (37). Histologically, the anal canal extends from the upper to the lower border of the internal anal sphincter and has, on formalin-fixed specimens, an average length of 3 cm (16). This slight difference in definitions and measurements must be accepted because the curved appearance of the anal verge cannot be evaluated histologically and because loss of tonus and the fixation procedures may influence the appearance of the specimens.

The anal lumen often forms a more or less triradiate slit when seen at anoscopy. The stem of the Y so formed approaches the midline posteriorly, and the arms embrace a pad of tissue anteriorly. Thus, three folds are seen, named the anal cushions (38). According to some investigators, the Y is asymmetric and usually embraces a pad of tissue to the right anteriorly (38,39). The anal cushions are present already in fetal life. In an opened surgical specimen from adults, these cushions are often inconspicuous.

The surface relief of the anal canal varies with age. The characteristic relief described in all anatomy textbooks is

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In adults (Figures 27.5, 27.9,27.10,27.11,27.12), the mucosa often shows less pronounced valves and columns and may exhibit a smooth surface with or without scattered anal sinuses and papillae (16).

The three epithelial zones are faintly visible to the naked eye on unstained, formalin-fixed specimens (Figure 27.5). The upper third shows the same relief as the mucosa of the rectum above. The area immediately above the anal valves and sinuses usually appears more smooth and gray-blue. This is the ATZ. Distal to the sinuses, the squamous epithelium appears as a smooth gray-brown area (Stroud's pecten). At the lower border of the anal canal, the dull, wrinkled perianal skin with hair follicles is obvious.

Musculature

The exact arrangement of the anal musculature is still a subject of debate, and new information is still being added using anal endosonography, magnetic resonance imaging, and manometry (40,41). The muscles here are described from the inside out (Figure 27.6).

Musculus Submucosae Ani

The musculus submucosae ani, originally described by Treitz (42), also have been called musculus sustentator tunicae mucosae, musculus mucosus ani, and musculus canalis ani (43). They consist of fibers from the intersphincteric longitudinal muscle, which pass through the internal sphincter, and from the internal sphincter itself. In the submucosa, they form a network around the vascular plexus (Figure 27.14) and are sometimes connected to the muscularis mucosae upward. Caudally, they rejoin the muscles from which they came. Some fibers fan out in the perianal skin, where they may join fibers from the corrugator cutis ani muscle (38,43).

Internal Anal Sphincter

The internal anal sphincter is a continuation of the circular muscle coat of the rectum but is considerably thicker. In histologic sections it measures 5 to 8 mm in thickness and ends 5 to 19 mm (average: 11.4) below the DL (16). Using anal endosonography, the thickness is maximally 4 to 5 mm (41). The muscle receives sympathetic (motor) fibers and parasympathetic (inhibitory) fibers from the inferior hypogastric plexus. In contrast to the adjacent rectum, the internal sphincter contains no enkephalin-immunoreactive fibers, few substance P reactive fibers, but moderate numbers of fibers reactive for neuropeptide Y and vasoactive intestinal peptide (44). Collagen fiber deposition increases with age (45) and is higher in patients with neurogenic fecal incontinence (46). Pelvic irradiation may result in increased fibrosis and nerve density (47).

Intersphincteric Longitudinal Muscle

The intersphincteric longitudinal muscle is situated between the internal and external sphincters (Figure 27.6). Its thickness varies, and its exact function is unknown (40,48). It contains unstriped fibers from the longitudinal muscle coat of the rectum and striped fibers from the levator ani muscles. At the lower end of the internal sphincter, it breaks up into a number of septae, which diverge fanwise through the subcutaneous part of the external sphincter to end in the corium, thus forming the characteristic corrugation of the perianal skin. These bands are, therefore, referred to as the corrugator cutis ani muscle.

External Sphincter

The striated anal sphincter is traditionally described as a circular muscle, which can be divided into a deep part and a superficial part that surround the internal sphincter and a subcutaneous part below these muscles (Figure 27.6). Most investigators have been unable to find a clear separation between these three parts (40). The puborectalis muscle is situated above the external sphincter. There are two prevailing theories about the arrangement of these muscles.

Shafik (49) has described three U-shaped loops, with their convexity directed backward, forward, and backward,

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The superficial and subcutaneous parts differ from normal striated musculature. They consist of relatively small muscle fibers, in the adult predominantly of the slow switch type (type 1), separated by delicate strands of connective tissue (51,52).

Blood Supply

The arterial supply to the anal canal is considerably greater than would be necessary for metabolism alone. Branches from the superior rectal artery reach the ATZ in nearly all cases, but the middle and inferior rectal arteries often also make substantial contributions (38).

The terminal branches of the arteries split up into small tortuous vessels, some of which form tiny arteriovenous anastomoses with the submucosal venous plexus. This plexus consists of small convoluted vessels with discrete dilatations, which may be fusiform, saccular, or serpentine. Connective tissue fibers and fibers from the musculus submucosae ani are situated between the vessels, thus anchoring them to the internal sphincter. The picture is more complex above the DL, whereas the dilatations are fewer and often larger below it. The drainage is mainly cranial to the superior rectal vein, but branches of the superior, middle, and inferior veins communicate freely below as well as through the internal sphincter (38).

Some investigators have described so-called anal glomeruli, in which groups of small convoluted vessels were surrounded by connective tissue, which separated them from the surrounding loose tissue (Figure 27.15). Such structures probably represent thrombosed vessels with recanalization. The whole system has been referred to as the corpus cavernosum recti (although it is situated in the anal canal) and is more pronounced in the areas corresponding to the anal cushions (39) (Figure 27.16).

Hemorrhoids

According to this new understanding of anal vasculature, hemorrhoids or hemorrhoidal disease should be defined as enlargement or prolapse of the anal cushions, in some cases followed by bleeding and thrombosis (53,54,55). However, it is still debated whether the term hemorrhoid or hemorrhoidal tissue also should be used to describe the normal anatomical structure or asymptomatic enlargement of the anal cushions (56,57).

The pathogenesis of symptomatic hemorrhoids has been debated for many years, and a low-fiber diet, constipation and prolonged straining, abnormal anal pressure, hypertension in the anal cushions, and hereditary and hormonal factors may be involved. Others have found hemorrhoids associated with diarrhea and obesity but not with age, constipation, cirrhosis, or varicose veins (58).

Deterioration of the supporting and anchoring connective tissue and degenerative changes of the collagen fibers, together with replacement of muscle tissue by collagen, which begins in the third decade, make the structures less stable. The result is venous stasis and dilatation and sliding down of the anal cushions, sometimes followed by prolapse. Damage to the surface may lead to bleeding of arteriolar origin. Inflammation and focal pressure may be

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This new understanding harmonizes well with the histologic picture of tissue from hemorrhoidectomies. The specimens most often consist of connective tissue stroma with scattered fibers of smooth muscle. The stroma may contain many blood vessels and larger vascular spaces that vary in size and configuration and are often less than 2 to 3 mm in diameter. Inflammatory changes and signs of recent or past thrombosis may be present, as may stromal fibromuscular hyperplasia and elastic fiber proliferation. In some cases the removed hemorrhoid more resembles a fibroepithelial polyp (54). Neuronal hyperplasia is common (60). The surface lining depends on the degree of descent of the cushion and of course the extent of the surgical excision. It therefore may show colorectal, ATZ, or squamous epithelium, as well as perianal skin. The mucosa may show inflammatory reaction or frank erosion or may be the site of regenerative, metaplastic, or keratotic reactions due to recurrent or permanent prolapse. The so-called fibrous anal polyp is assumed to represent an end stage of a prolapsed cushion (61).

Other Vascular Changes

There are a few differential diagnoses to chronic hemorrhoidal disease. Perianal thrombosis, formerly referred to as perianal hematoma, is an acute dark hard lesion arising under the squamous zone of the anal canal or the perianal skin. It represents a thrombus lying in the larger venous dilatations (61,62). Rectal varices are a result of portal hypertension and represent dilated submucosal veins formed when the communicating veins that connect the superior with the middle and inferior hemorrhoidal veins become enlarged. They extend above the level of hemorrhoids and occasionally downward to reach the buttock, perineum, or upper thigh (63,64). Diffuse cavernous hemangioma of the rectosigmoid is a vascular malformation consisting of vascular channels of variable size and thickness, which may involve the whole bowel wall and mesentery or perirectal tissue and may extend down to the DL (65). Recently, two cases of Dieulafoy's lesion in the anal canal have been described (66).

The vast majority of anal fissures occur in the posterior midline. A variant of the course of the inferior rectal artery has been blamed for a reduced perfusion of the posterior commissure and thereby of significance in the pathogenesis (67). Using Doppler laser flow cytometry, it has been shown that the blood flow in the posterior quadrant of the anal canal and perianal skin is significantly lower than in the anterior left and right quadrant and that the higher the anal pressure, the lower the perfusion (68). In addition, histologic studies have shown a lesser arteriolar density in the posterior quadrant throughout the anal canal (69). These observations have led to the hypothesis that anal fissures are ischemic ulcers.

Nerves

Ganglion cells in the rectum are found in three separate plexuses: the superficial and deep submucous and the myenteric. In the anal canal, ganglion cells belonging to these plexuses are absent or sparse in the first centimeter above the DL (70). The diagnosis of ultrashort Hirschsprung's disease can be made by finding increased acetylcholinesterase reaction in muscularis mucosae and submucosa immediately above the DL (71). The density of interstitial cells of Cajal is significantly lower in the internal sphincter than in the circular muscle layer of the rectum (72).

The sensory innervation (73) of the lower part of the anal canal is accomplished by the inferior rectal nerves. The lining contains free nerve endings as well as organized endings (Meissner's corpuscles, Krause's end bulbs, Golgi-Mazzoni bodies, and genital corpuscles). It is sensitive to touch, pain, heat, and cold.

Sensitivity also can be demonstrated just above the DL in the ATZ, where the same endings can be found just above the anal valves. Thus, pain occasionally can be felt from injections given just above the DL. The ATZ is well supplied with intraepithelial nerve fibers and endings (74). Both types of nerve endings cease abruptly at the transition to the colorectal zone, where only a poorly defined dull sensation is present. However, discrimination seems not to be impaired by excision of the ATZ after restorative proctocolectomy (75).

Patients with hemorrhoids have a mild anal sensory deficit, probably due to the mucosal descent (76). Surgical specimens of hemorrhoids and their end stage, the fibrous polyp, may show proliferation of peripheral nerves without increased terminal density (60) (Figure 27.17).

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Lymphatics

Lymphoid follicles are present in the anal canal from the DL and upward in approximately the same number as in the rectum (about 25/cm²) (77).

The lower part of the anal canal (below the DL) and the perianal skin are drained to the superficial inguinal nodes. The drainage of the upper part is more complex, and varying results have been obtained from injection studies. Most investigators agree that an indirect or intramural system connects lymphatics in the anal canal to the lymphatic plexuses of the rectum (78). Other more direct lymphatics follow the inferior or medial rectal vessels to end in the hypogastric, obturator, and internal iliac nodes, or they follow the superior rectal vessels to end in nodes in the sigmoid mesocolon and near the origin of the inferior mesenteric artery. Occasional connections also have been demonstrated to the common iliac, middle and lateral sacral, lower gluteal, external iliac, and deep inguinal nodes (79).

Lymphatic metastases from carcinoma of the anal canal are most often found in the inguinal, femoral, pararectal, and iliac nodes, and eventually in the mesenteric or para-aortical nodes. Hematogenous metastases are most often located in the liver and lungs (31,36). Studies on sentinel nodes in anal cancer are ongoing (80).

Histology

Colorectal Zone

The mucosa of the upper, colorectal zone is a continuation of the rectal mucosa, and no line of demarcation marks the transition to the anal canal. However, some shortening and irregularity of the crypts often are found in the area closest to the ATZ (Figure 27.18).

Anal Transitional Zone and Anal Glands

As stated above, the upper border of the ATZ is defined by the appearance of other epithelial types. In one-third of cases, the upper part of the ATZ consists of a small rim of mature squamous epithelium (Figures 27.8, 27.18) (16). Many epithelial variants can be found in the ATZ, the most prominent being the so-called ATZ epithelium.

The term ATZ epithelium (81) is not generally accepted but can be used until an exact classification is established. It consists of four to nine cell layers. The basal cells are small, and their nuclei are arranged perpendicular to the basement membrane. The surface cells can be columnar (Figure 27.19), cuboidal or polygonal (Figure 27.20), or flattened (Figure 27.21). Small areas can show umbrella-shaped surface cells and more distinct cell borders (Figure 27.22). Other epithelial types are often present in the ATZ. Small areas of mature squamous epithelium may be present, especially at the upper border. Scattered crypts of colorectal type and small areas of simple columnar epithelium also may be found.

The anal (intramuscular) glands open in the ATZ (Figure 27.8). These glands seem to be present in all anal canals and the median number is six, with a range of three to ten. Four of five are only present in the submucosa, but the rest extend into the internal sphincter and a few percent reach the intersphincteric space or even penetrate the external sphincter (82,83). The epithelial lining and mucin production are similar to the ATZ epithelium (84), and endocrine cells are occasionally present (85). A characteristic feature is the presence of intraepithelial microcysts and occasionally goblet cell metaplasia (Figure 27.23). The epithelium is surrounded by one or two cell layers of myoepithelial cells that stain positive for smooth muscle actin (83). The glands are often surrounded by groups of lymphocytes. It is

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Squamous Zone

The transition to uninterrupted squamous epithelium usually takes place at the level of the DL. The squamous epithelium is unkeratinized with short or no papillae. Melanocytes increase in number as the perianal skin is approached, and the epithelium contains dendritic (Langerhans) cells (60) (Figure 27.24), T lymphocytes (87), and Merkel cells (88) (Figure 27.25). These cells also may be present in squamous metaplasia covering hemorrhoids. The number of dendritic cells is increased in cases of condylomata but decreased in case of concomitant HIV infection (89). Glands or skin appendages are never present in the squamous zone.

Perianal Skin and Glands

Keratinization becomes apparent at the lower end of the anal canal, and the squamous zone gradually merges into the perianal skin with sweat glands, hairs, and sebaceous glands. A characteristic finding is the presence of apocrine glands in the subcutaneous tissue (Figure 27.26). An additional type of glands, the so-called anogenital sweat glands, are lined by a simple columnar epithelium with cytoplasmic snouts protruding into the lumen and resting on a layer of flat myoepithelium (90) (Figure 27.27). These glands are probably the point of origin for papillary hidradenoma (91).

Lamina Propria and Muscularis Mucosae

The lamina propria consists of loose connective tissue which in the squamous zone often contains a variable number of

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The well-defined muscularis mucosae of the rectum continue in the colorectal zone of the anal canal (Figure 27.18) and can be found in the upper part af the ATZ (Figure 27.14). Fibers extending up between colorectal crypts indicate mucosal prolapse, and most fibroepithelial polyps also contain fibers from the muscularis mucosae (92,94).

Special Techniques

Mucin Histochemistry

The columnar variant of the ATZ epithelium produces small amounts of a mixture of sulfomucins and sialomucins,

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Cytokeratins

The keratin expression of the various epithelial types in the anal canal is complex (96). Briefly, the typical reactions in colorectal type epithelium are CK20+/CK7 and in ATZ epithelium and anal glands CK7+/CK20 , and this pattern is also found in tumors originating in the respective epithelia (86,97,98).

The reaction for CK7 varies in the ATZ epithelium, sometimes being very strong throughout all layers, sometimes being absent in the basal layer or the whole epithelium (Figure 27.29). In addition, the ATZ and anal glands are usually positive for cytokeratins 4, 8, 13, 16, 17, 18, and 19. The coexpression of CK4 and CK13 has also been observed in the transitional epithelium of the urinary bladder.

Proliferation Markers

The proliferative marker MIB-1 for Ki-67 is positive in a layer of cells immediately above the basal layer and may be quite pronounced, especially in cases of hemorrhoids and prolapse (Figure 27.30). However, the reaction still differs from that seen in anal intraepithelial neoplasia (Figure 27.31).

Hormone Receptors

Androgen, estrogen, and, to a lesser degree, progesterone receptors have been demonstrated in the squamous epithelium, as well as in the underlying stroma and in the smooth muscle cells of the internal sphincter in most individuals and at all ages, indicating that the organ is a target for sex steroid hormones (99). The anogenital sweat glands and the corresponding papillary hidradenoma show positive reaction for estrogen and progesterone receptors, in contrast to conventional sweat glands (91) (Figure 27.32).

Neuroendocrine Markers

The characteristic epithelia of the ATZ and anal glands contain a heterogeneous population of endocrine cells (81,85), which all show positive reaction for synaptophysin and chromogranin A (Figure 27.33). The cells have slender processes that approximate the surface and a fine granular cytoplasm. The number varies, and the largest population also reacts for serotonin and pancreastatin, but cells positive for somatostatin, peptide tyrosine tyrosine, glucagon-like peptide 1, calcitonin gene related peptide, protein gene product 9.5, and neurotensin are occasionally present. The pattern is a mixture of products typical for colorectal epithelium and for Merkel cells in the squamous epithelium (88,100).

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Melanin-Containing Cells

A few melanocytes are also occasionally present in the ATZ (85,101,102). These are CK7 /CK20 and positive for S-100 protein, Melan A, and HMB-45 (Figure 27.34). The corresponding malignant melanoma may be amelanotic and positive for polyclonal but not monoclonal carcinoembryonic antigen (CEA) (103).

Flow Cytometric Microscopy and Electron Microscopy

Flow cytometric DNA analyses of the ATZ epithelium have shown a dominating diploid population with a small hyperdiploid peak, resembling the picture of metaplastic epithelium (104). Transmission electron microscopy has not shown asymmetric unit membranes. In scanning electron microscopy, the surface cells show distinct cell borders and short microvilli (Figure 27.35) (81).

Metaplasia and Heterotopia

Other epithelial types may be present and sometimes indicate pathologic conditions. Occasionally, colorectal-type crypts in the ATZ may contain Paneth's cells (Figure 27.36) or show pyloric metaplasia (Figure 27.37), probably as a result of mucosal damage (as in longstanding colitis). Squamous metaplasia in the ATZ and even in the colorectal zone is seen in cases of prolapse, and keratinization may be present (Figure 27.38).

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I have seen a single example of fundic gastric mucosa in the anal canal. Such areas, rarely found in the rectum, are histochemically almost identical to their orthotopic counterpart (105). Probably Helicobacter pylori can turn up in the anal canal as it has done in body-type gastric epithelium in the rectum (106). A unique case has been reported showing ectopic prostatic tissue in the lower anal canal (107).

Viral and Neoplastic Changes

Cytomegalovirus, which occasionally infects the rectum, is rarely seem in the anal canal histiocytes (108). Herpesvirus-induced changes are more commonly found in the squamous epithelium (Figure 27.39), and koilocytotic changes due to human papillomavirus (HPV) can be found as high up as in the ATZ (Figure 27.40).

Anal intraepithelial neoplasia (AIN), also referred to as dysplasia or anal squamous intraepithelial lesion (ASIL), is not uncommon. It may be present in the squamous zone as well as in the ATZ and in minor surgical as well as in resection specimens (Figures 27.31, 27.41) (109,110). Squamous carcinoma and AIN are particularly common among men who have sex with men, patients with HIV/AIDS, and transplant recipients and in the rare syndromes Wiskott-Aldrich and epidermodysplasia verruciformis, indicating an attenuated immune response is of importance (111). High-risk HPV is found in most lesions, in particular those located high in the anal canal (112). Exact and reproducible grading of AIN is difficult in biopsies, as well as in cytologic specimens (113,114,115). Follow-up studies have shown that many cases of AIN I II will regress but that a considerable number of AIN III will recur, even aften highly active antiviral therapy. There are however only few documented cases of transition to

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Squamous carcinoma of the anal canal may have different appearances (i.e., basaloid or keratinizing), but there is substantial overlap of the histologic features found in these tumors (112,118), and the reproducibility of the older WHO classification is rather low (119). In the recent WHO classification, it is therefore recommended that the generic term squamous carcinoma be used for these tumors (31). Prognostic markers have not yet been identified (120). A summary of tumors originating in the different zones is listed in Table 27.3.

Finally, it should be remembered that the anal area is a common site of extramammary Paget's disease and that many of these patients have a synchronous or metachronous malignant tumor. Paget's cells must be distinguished from artifactual clear cells, glycogen-rich cells, koilocytes, and clear cells in pagetoid dyskeratosis, pagetoid Bowen's disease and pagetoid melanoma (121) (Figure 27.42).

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Function

The normal function of the rectum and anal canal is the result of a combination of factors, including not only the puborectalis and sphincter muscles, but also stool volume and consistency, rectal storage capacity, sensation, and delivery, as well as cognitive and behavioral influences. The functional disorders, the various techniques for investigation of anorectal dysfunction, and the diagnosis and treatment have been extensively reviewed recently (123,124,125). The significance of the ATZ for normal anal function is still a subject of debate (126).

Gender and Aging

There is little information on differences in the anal canal between the two sexes and as a result of aging. The surgical anal canal is a few millimeters shorter in females (4.4 cm

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The mucosal surface is often more smooth in the elderly, and the papillae may be more pronounced (Figure 27.5). The underlying connective tissue becomes less stable, which may result in descent of the anal cushions and subsequent squamous metaplasia of the ATZ (54). The number of lymphoid follicles does not change with age (77).

Specimen Handling

In minor surgical specimens it is often possible to identify the smooth gray-brown squamous zone from the gray-blue, more glistening anal mucosa. The tissue always should be cut and embedded along the longitudinal axis in order to describe the lesions (i.e., AIN) in relation to the different epithelial zones.

Local excisions should be provided by the surgeon with a suture at the proximal end, pinned up on a plate and sectioned in the same way with special attention to resection lines. Larger surgical specimens are rare nowadays where anal carcinoma most often is treated nonsurgically. They should also be pinned up, and multiple sections may be necessary to find or exclude residual tumor tissue. Information on previous radiation therapy should be provided.

Normally most diagnoses can be established without the use of special stains, but occasionally a panel of immunohistochemical reactions is useful (Table 27.4).

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