Diagnosis, follow up, and treatment of anal intraepithelial neoplasia are complex and not standardized. This may be partly caused by poor communication of biopsy and cytology findings between pathologists and clinicians as a result of a disparate and confusing terminology used to classify these lesions. This article focuses on general aspects of epidemiology and on clarifying the current terminology of intraepithelial squamous neoplasia, its relationship with human papilloma virus infection, and the current methods that exist to diagnose and treat this condition.
Key points
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AIN can be managed by close observation, topical treatments, photodynamic therapy and surgery.
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Surgical approaches for AIN include anal mapping with wide local excision with or without flaps, or more limited removal using targeted destruction with high-resolution anoscopy.
Introduction
Although anal squamous cell carcinoma (SCC) remains an uncommon malignancy its occurrence has been steadily rising in the United States. Previous to the AIDS epidemic the incidence reported was of 0.7 per 100,000 individuals ; subsequently the rate rose to approximately 2 per 100,000. Extensive research conducted during the last decades has shown that this disease is more closely related to genital rather than to gastrointestinal malignancies. Historically women have been more frequently affected by these tumors than males; however, newer studies have revealed similar rates between the two genders. It has been postulated that the higher proportion of anal SCC is linked to an increased number of lifetime sexual partners for both men and women, changing sexual practices including both women and men engaging in anoreceptive intercourse, and an increased number of men having sex with men (MSM). Similarly to colonic malignancies in which invasive adenocarcinoma evolves from premalignant lesions (adenoma-carcinoma sequence), anal SCC progress through a continuum of preinvasive lesions known as anal intraepithelial neoplasia (AIN). Timely diagnosis of AIN is therefore of paramount importance to lower the incidence of invasive carcinoma, which frequently carries a very poor prognosis. Diagnosis, follow-up, and treatment of AIN is complex and not standardized, which may be partly related to poor communication of biopsy and cytology findings between pathologists and clinicians as a result of a disparate and confusing terminology used to classify these lesions. This article focuses on general aspects of epidemiology and on clarifying the current terminology of intraepithelial squamous dysplasia, its relationship with human papilloma virus (HPV) infection, and the current methods used to diagnose and treat this condition.
Introduction
Although anal squamous cell carcinoma (SCC) remains an uncommon malignancy its occurrence has been steadily rising in the United States. Previous to the AIDS epidemic the incidence reported was of 0.7 per 100,000 individuals ; subsequently the rate rose to approximately 2 per 100,000. Extensive research conducted during the last decades has shown that this disease is more closely related to genital rather than to gastrointestinal malignancies. Historically women have been more frequently affected by these tumors than males; however, newer studies have revealed similar rates between the two genders. It has been postulated that the higher proportion of anal SCC is linked to an increased number of lifetime sexual partners for both men and women, changing sexual practices including both women and men engaging in anoreceptive intercourse, and an increased number of men having sex with men (MSM). Similarly to colonic malignancies in which invasive adenocarcinoma evolves from premalignant lesions (adenoma-carcinoma sequence), anal SCC progress through a continuum of preinvasive lesions known as anal intraepithelial neoplasia (AIN). Timely diagnosis of AIN is therefore of paramount importance to lower the incidence of invasive carcinoma, which frequently carries a very poor prognosis. Diagnosis, follow-up, and treatment of AIN is complex and not standardized, which may be partly related to poor communication of biopsy and cytology findings between pathologists and clinicians as a result of a disparate and confusing terminology used to classify these lesions. This article focuses on general aspects of epidemiology and on clarifying the current terminology of intraepithelial squamous dysplasia, its relationship with human papilloma virus (HPV) infection, and the current methods used to diagnose and treat this condition.
Historical perspective
Although premalignant and malignant lesions of the anus and perianal region had long been described, it was not until 1962 when Turell recognized that anal skin and anal canal SCC were histologically and biologically distinct lesions. Turell realized that perianal tumors often displayed keratinization when examined histologically and were associated with a favorable clinical course; SCC of the anal canal, however, usually exhibited poor histologic differentiation and often metastasized to inguinal and pelvic lymph node metastasis. Subsequently, a possible etiologic relationship between HPV, anal warts, and in situ and invasive SCC was not proposed until 1971 by Oriel and Whimster who used electron microscopy for the identification of HPV particles on these lesions.
Fenger and Nielsen described dysplasia for the first time in 1981. The same authors in a relatively small (19 patients) but revealing study linked anal and uterine cervix neoplasia and proposed adoption of a similar terminology to the already existing cervical intraepithelial neoplasia (CIN) as anal canal intraepithelial neoplasia I–III.
More recently extensive research has provided conclusive evidence between the association of HPV and AIN and the link between certain HPV types and the likelihood of developing high-grade squamous dysplasia and invasive carcinoma The rise of the AIDS epidemic has further advanced the understanding of the risk factors associated with the development of this disease and its natural history and those clinicopathologic factors that influence clinical progression.
Epidemiology and incidence
The exact incidence of AIN is difficult to estimate. Impaired cellular-mediated immunity, as found in patients with AIDS and organ transplant recipients, is a strong risk factor for low- and high-grade AIN. The relationship between HPV-driven disease and the host immune defense mechanisms is complex and numerous factors besides an adequate CD4 count play a role in the natural progression of the infection.
The iatrogenic immunosuppression that follows organ transplantation has been shown to increase the incidence of high-grade dysplasia and anal cancer 10- to 100-fold compared with the general population, especially in women, who are affected twice as often as men. Most of these data have been compiled from patients receiving drugs that typically elicit a profound dysfunction of the cellular arm of the immune system (calcinurin inhibitors, azathioprin). Current studies are being conducted to assess the impact of newer medications that exert a more selective immunomodulation and may therefore be associated with lower risk of developing high-grade AIN and anal cancer.
The frequency of anal cancer and AIN is significantly increased in men and women infected with HIV independent of the route by which the virus was acquired. It is, however, especially prevalent in MSM, who are 20 times more likely to develop this disease than heterosexuals. HIV-positive MSM carry the highest incidence of HPV-related disease including high-grade AIN and invasive SCC. In a meta-analysis conducted in Australia the pooled prevalence of high-grade AIN and invasive SCC was 29.1% and 21.5% and 45.9 per 100,000 and 5.1 per 100,000 in HIV-positive MSM versus HIV-negative MSM, respectively. Furthermore, the detection of HPV-16 and -18 was much higher in HIV-positive MSM than in HIV-negative MSM. HIV-infected women have a nearly sevenfold increased risk of anal cancer compared with the general population.
There is a clear but not fully characterized relationship between these two sexually transmitted viruses, HIV and HPV. It has been shown that HPV infection is more likely to persist in HIV-positive patients and to evolve in a more aggressive clinical fashion possibly related to the inadequate cellular immunity, which is critical in the clearance of viral organisms. Infection with multiple types of HPV is also more commonly noted in HIV-positive patients. It has also been shown that HPV infection itself increases the risk of acquiring HIV. In a study conducted in the United States, a cohort of 1409 MSM with HPV disease had a significantly higher chance of HIV seroconversion than in HPV-negative individuals (hazard ratio, 3.5; 95% confidence interval, 1.2–10.6).
Predictive factors for progression to high-grade AIN and invasive carcinoma
There are no solid data on the rate of progression regarding low- to high-grade AIN and invasive carcinoma. Two studies with a follow-up of almost two decades have revealed that overall 5% of high-grade AIN evolves into invasive carcinoma. In a study of HIV-positive and HIV-negative MSM, Palefsky and coworkers found a rate of progression from low- to high-grade AIN in 62% and 36% of patients, respectively. These findings were similar to another study of 41 HIV-positive MSM in whom normal epithelium or low-grade AIN evolved into high-grade AIN over a 17-month period.
The presence of high-risk viral phenotypes, multiple HPV-type infections, low levels of CD4 lymphocytes, and smoking have all been suggested as factors that accelerate progression to high-grade AIN and invasive carcinoma.
The use of highly active antiretroviral therapy in patients with HIV has dramatically decreased the incidence of most defining AIDS cancers. However, the incidence of high-grade AIN and anal cancer have not been affected by the introduction of highly active antiretroviral therapy. The explanation for this observation remains unknown.
The natural history of AIN progression in HIV-negative heterosexual men and women is much less understood and solid data addressing this issue are lacking. In a large ongoing study in 431 women with follow-up at the 4-month interval, it was shown that 50% had incidental anal HPV, which was sequentially acquired after cervical infection. Interestingly, in 87% of these patients the infection resolved within 1 year, which is much shorter than the approximately 2-year duration typically reported for elimination of cervical HPV.
HPV and its role in AIN and invasive SCC
The similarities between anal and cervical preinvasive and invasive squamous lesions can be partly explained by the anatomic and histologic resemblances between these two organs. That is, both have a transformation zone composed of a squamous-columnar epithelial junction in which the columnar epithelium of the lower rectum commonly undergoes squamous metaplasia. Most AIN and invasive carcinoma arise within this area. There is compelling scientific evidence that HPV is the cause of premalignant and malignant squamous tumors of the lower anogenital tract including uterine, cervix, vagina, vulva, anus, perianal skin, and scrotum. Numerous molecular virology studies performed in cell lines and in human tissue have demonstrated the capability of HPV to induce transformation from nonneoplastic epithelium to AIN1 to AIN 3 and finally invasive carcinoma.
Human papilloma are small double-stranded DNA viruses with an exquisite predilection for squamous epithelium. Currently, more than 130 HPV types have been characterized and within the different types some viruses show specific tropism for either cutaneous or mucosal tissue. Anogenital infection is usually linked to a group of 30 to 40 types that can be divided into those predominantly associated with indolent, self-limited disease, including condylomas, AIN1, and, CIN1 (subtypes 6, 11, 42, 43, and 44) and those most commonly isolated from high-grade lesions and invasive carcinomas (subtypes 6, 18, 31, 33, 35, 39, 45, 50, 51, 55, 56, 58, 59, and 68). Understanding the differences in the potential to induce malignant transformation among the different types of HPV has been instrumental in guiding follow-up and treatment in affected men and women. Furthermore, in 2008, Harald zur Hausen was awarded the Nobel Prize for his discovery of the role HPV types 16 and 18 play in the development of cervical cancer. The association between high-risk HPV types and cervical cancer is robust with very strong odds ratios for HPV 16 and 18 and tight confidence limits.
HPV prevalence in high- and low-grade AIN has been reported at 91% and 88%, respectively. HPV types 16 and 18 are the most commonly identified in high-grade dysplasia and invasive SCC (72% and 69%, respectively). Contrary to low-risk HPV types that remain in the host cells as a plasmid, high-risk types become integrated into the keratynocyte leading to overexpression of oncogenes and inhibition of tumor-suppressor genes and DNA aneuploidy. This state of genetic chaos allows the progression from low- to high-grade dysplasia and invasive carcinoma.
The evolution of the nomenclature and grading
Historically, grading of the dysplastic changes within the anal canal squamous mucosa was based in great part on the subjective opinion of the examining pathologist and classified into three categories: (1) mild, (2) moderate, or (3) severe. These three categories were then renamed AIN1, 2, and 3 extrapolating the data that existed on the uterine cervix (CIN1, 2, and 3) and its association with HPV. This approach was supported by the fact that the uterine cervix and the anal canal have common embryologic pathways. Studies on cervical lesions highlighted that CIN1 and 3 had the lowest interobserver variability among pathologists, but CIN2 lacked consistency among observers. This poor reproducibility was also demonstrated for the anal region, and thus the term AIN2 or moderate dysplasia was interpreted by clinicians as equivocal for the possibility of a preneoplastic lesion. This uncertainty made it difficult among clinicians to decide on a specific therapy. It became evident that this intermediate category lacked biologic correlation because it merely combined a low-grade lesion with a premalignant disease, which cannot be accurately distinguished on hematoxylin and eosin stained sections. This observation explained the poor reproducibility among observers. Further complicating the issue, cases formerly diagnosed as moderate dysplasia (grade 2) turned out to be severe dysplasia on follow-up.
Another point of potential confusion is that the American Joint Committee on Cancer promoted the term squamous intraepithelial lesion (SIL) either as low or high grade, whereas the World Health Organization still recommended the use of AIN terminology low- and high-grade categories. SIL and CIN therefore described the same types of lesions and could be interchangeable.
With advancements made in the biology of HPV the grading was modified to reflect the behavior of the HPV infection and its effects on the anal squamous mucosa, and the term squamous intraepithelial lesion was introduced with a two-tier grading system: low grade corresponding to AIN1 and high grade to AIN2 and AIN3.
The pathology community recognized the need to standardize the nomenclature related to this topic to avoid potential serious misunderstandings in the communication of pathology results. There was a clear need to unify the concepts of SIL and AIN, and to modify the grading system in a way that it would better guide treatment. The proposal is to abandon dysplasia grade 2 not only to follow the route established by the Bethesda grading for cytology, but more importantly to apply the knowledge propelled by several advances in molecular pathology that shed light into the natural history of HPV infection and further strengthened the concept of a two-tiered grading system for squamous dysplasia.
It is now known that HPV infection can take two behavior routes: either that of a transient infection in which the virus replicates in an active but self-limited manner; or in the form of a persistent carrier state, which is associated with the development of carcinoma. In this regard, the grading of AIN can then be classified into two-tier, low-grade SIL (LSIL), which reflects a transient, self-limited infection, and high-grade SIL (HSIL), which is associated with chronic and long-standing infection. Expression of biomarkers by immunohistochemistry, such as p16, is in keeping with this two-tier grading system wherein the HSIL cases stain strongly and consistently and the LSIL stain negative. This immunoprofile further supports the two- versus a three-tier system. Immunohistochemical stain for p16 is an excellent tool to discriminate between an indolent lesion (transient infection) and one associated with carcinoma (persistent infection).
The two-tier system of LSIL and HSIL is similar to that for other genital areas including the cervix, vagina, vulva, perianus, and penis. Ideally, a uniform, reproducible, and standardized histologic evaluation would be applied to all these sites to reduce diagnostic variability among pathologists and thus facilitate accuracy for research purposes. Based on this premise and with the goal of obtaining unity in the definitions of dysplasia, in 2012 the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology through the work of approximately 60 physicians from various specialties who are experts in anogenital disease sponsored standardization of the terminology for HPV-related lesions affecting the epithelium of the anal canal. The recommendation of using a two-grade system was also endorsed by numerous other professional societies and government agencies. This support gave the current nomenclature an “official” connotation: LSIL and HSIL ( Figs. 1 and 2 ).
Pathology of AIN
Anal intraepithelial HPV-associated squamous lesions are classified as LSIL and HSIL. The gross appearance of AIN is polymorphic because the lesions may present as plaques, papules, eczema, or papilloma-like areas. The presence of induration or ulceration may be seen when the lesion is an invasive carcinoma.
Microscopically, the changes are limited to the epithelium where there is increased cellularity accompanied by nucleomegaly, pleomorphism, increased nuclear to cytoplasmic ratio, irregular nuclear contours, and denser chromatin. In LSIL the changes predominately effect the lower third of the full epithelial thickness, with maturation starting at the middle third, leaving the upper third close to normal. Although mitosis may be observed, abnormal mitotic figures and marked nuclear atypia are not characteristic of LSIL. With the presence of HPV cytopathic changes, such as koilocytosis, multinucleation, nuclear irregularity, and perinuclear clearing of the cytoplasm, may be present, but these features do not correspond to HSIL. The mere presence of HPV cytopathic changes, such as koilocytosis, in the absence of high-grade lesion should be classified as a LSIL.
For HSIL, in addition to the findings of LSIL, there is lack of maturation in the upper two-thirds of the epithelium. Mitotic figures, typical or abnormal, are also present in the middle or the upper third of the epithelium. Abnormal mitosis and marked nuclear atypia are not characteristic of an LSIL, but may be present in a sample with maturation starting at the middle third and upward creating diagnostic uncertainty. In this situation a strong positive stain for p16 facilitates diagnosis of HSIL.
Another cause for diagnostic uncertainty arises when the sample is distorted, particularly as a result of the cauterization effect on tissues. In this situation, the distortion makes evaluation of the dysplastic lesion difficult to distinguish between low- and high-grade lesions. Immunostaining for p16 may be helpful in this situation. Figs. 3 and 4 show HSIL, which involves the transition zone and extends into the proximal colonic mucosa.
Another difficult scenario is when, in an otherwise low-grade lesion or the presence of condyloma, there are focal areas that raise the possibility of a proximal high-grade lesion as seen in Figs. 5 and 6 . Immunostaining for p16 is helpful to exclude the presence of HSIL. As seen in the photomicrograph negative p16 not only distinguishes tangentially cut basal cells from HSIL, but it helps to separate LSIL, immature squamous metaplasia, atrophy, and regenerative/reactive changes. The staining in these lesions is either absent or only focal. True diagnostic stain for HSIL should be diffuse and strong in the nuclei alone or in both nuclei and cytoplasm.
